Methods and compositions for labeling polypeptides

ABSTRACT

Synthesis of many proteins is tightly controlled at the level of translation and plays an essential role in fundamental processes such as cell growth and proliferation, signaling, differentiation or death. Methods that allow imaging and identification of nascent proteins allow for dissecting regulation of translation, both spatially and temporally, including in whole organisms. Described herein are robust chemical methods for imaging and affinity-purifying nascent polypeptides in cells and in animals, based on puromycin analogs. Puromycin analogs of the present invention form covalent conjugates with nascent polypeptide chains, which are rapidly turned over by the proteasome and can be visualized and specifically captured by a bioorthogonal reaction (e.g., [3+2] cycloaddition). The methods of the present invention have broad applicability for imaging protein synthesis and for identifying proteins synthesized under various physiological and pathological conditions in vivo.

RELATED APPLICATION

The present application is a continuation of and claims priority under35 U.S.C. §120 to U.S. application, U.S. Ser. No. 13/673,296, filed Nov.9, 2012, which claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application, U.S. Ser. No. 61/558,107, filed Nov. 10,2011, each of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The entire set of cellular proteins is generated through translation ofmRNAs by ribosomes. The identity and amount of the proteins that a cellsynthesizes are critical parameters in determining the physiologicalstate of the cell. Protein synthesis is frequently not proportional tomRNA levels, mainly because translation is often tightly regulated;indeed, many critical controls in gene expression occur at the level oftranslation (Sonnenberg et al., Cell 136(4):731-745 (2009); Selbach etal., Nature 455(7209):58-63 (2008); Baek et al., Nature 455(7209):64-71(2008)). Under specific conditions (such as heat shock, starvation,availability of iron, etc.), translational controls ensure thatsynthesis of specific cellular proteins is quickly turned on or off.Translational controls are particularly prominent in systems in whichtranscription is inhibited, such as in early embryonic developmentbefore the onset of zygotic transcription. Furthermore, translation ofmany proteins is spatially localized, as underscored by the finding thatthe majority of mRNAs in Drosophila embryos display distinct subcellularpatterns (Lecuyer et al., Cell 131(1):174-187 (2007)).

Understanding how gene expression is regulated at the level oftranslation, spatially and temporally, requires tools for visualizingand identifying nascent polypeptide chains. The current method used forthis purpose relies on the biosynthetic incorporation of azide- oralkyne-bearing methionine (Met) analogs such as azidohomoalanine (Aha)(Dieterich et al., Proc. Natl. Acad. Sci. USA 103(25):9482-9487 (2006);Link et al., J. Am. Chem. Soc. 125(37):11164-11165 (2003)) orhomopropargylglycine (Hpg) (Beatty et al., Angew. Chem. Int. Ed. Engl.41(14):2596-2599 (2002)). The resulting azide or alkyne-labeled proteinscan be detected by copper(I)-catalyzed azide-alkyne cycloaddition(CuAAC) (Rostovtsev et al., Angew Chem. Intl. Ed. Engl. 41(14):2596-2599(2002); Tornoe et al., J. Org. Chem. 67(9):3057-3064 (2002); Wang etal., J. Am. Chem. Soc. 125(11):3192-3193 (2003)) with reagents forfluorescence detection (Beatty et al., Angew. Chem. Int. Ed. Engl.41(14):2596-2599 (2002)) or for affinity purification and identificationby mass spectrometry (Dieterich et al., Proc. Natl. Acad. Sci. USA103(25):9482-9487 (2006)). Though simple and robust, this method has anumber of drawbacks. Cells prefer Met over Aha or Hpg by a factor ofabout 500 (Beatty et al., Angew. Chem. Int. Ed. Engl. 41(14):2596-2599(2002)), which means cultured cells need to be labeled with Aha or Hpgin Met-free media; this limitation precludes the use of Aha and Hpg tostudy protein synthesis in whole animals. To be incorporated intoproteins, Aha and Hpg need to be activated as aminoacyl-tRNAs, a stepwhich limits the temporal resolution of this method. Finally, thismethod generates full-length Aha- or Hpg-labeled proteins, not nascentpolypeptide chains. Improved labeling techniques are needed for thestudy of nascent proteins in vivo, in particular methods that are rapid,sensitive, and work well in whole organisms.

SUMMARY OF THE INVENTION

Synthesis of many proteins is tightly controlled at the level oftranslation and plays an essential role in fundamental processes such ascell growth, proliferation, signaling, differentiation, and death.Methods that allow imaging and identification of nascent proteins allowfor studying regulation of translation, both spatially and temporally,including in whole organisms. Described herein are robust chemicalmethods and systems for imaging and affinity-purifying nascentpolypeptides in cells and in animals based on puromycin analogs.Puromycin analogs of the present invention form covalent conjugates withnascent polypeptide chains, which are rapidly turned over by theproteasome and can be visualized and specifically captured by abioorthogonal reaction (e.g., [3+2] cycloaddition, Staudinger ligation,tetrazine ligation). The methods of the present invention have broadapplicability for imaging protein synthesis and for identifying proteinssynthesized under various physiological and pathological conditions invivo and in vitro.

In one aspect, the present invention provides puromycin analogs ofFormula (I):

or a salt thereof, wherein R^(A), R^(B), R¹, R^(1′), R², R³, R⁴, R⁵, andR⁶ are as defined herein.

In some embodiments, a puromycin analog according to the presentinvention is of Formula (II):

or a salt thereof, wherein R^(A), R¹, R², R³, R⁴, R⁵, and R⁶ are asdefined herein.

In some embodiments, a puromycin analog according to the presentinvention is of Formula (III):

or a salt thereof, wherein R^(A), R¹, R², R³, R⁴, and R⁵ are as definedherein.

In some embodiments, a puromycin analog according to the presentinvention is of Formula (III-a):

or a salt thereof, wherein R^(A) and R¹ is as defined herein.

In certain embodiments, the present invention provides

In another aspect, the present invention provides methods of labelingpolypeptides. In certain embodiments, a method of the present inventionincludes a method of labeling a polypeptide comprising providing apolypeptide-puromycin analog conjugate comprising a first reactivegroup; and contacting the polypeptide-puromycin analog conjugate with acompound comprising a second reactive group and a label, such that abioorthogonal reaction occurs between the first and second reactivegroups. In certain embodiments, a method of the present inventionincludes a method of labeling a polypeptide comprising providing apolypeptide-puromycin analog conjugate comprising a first reactiveunsaturated group; and contacting the polypeptide-puromycin analogconjugate with a compound comprising a second reactive unsaturated groupand a label, such that a [3+2] cycloaddition occurs between the firstand second unsaturated groups. In certain embodiments, the firstreactive unsaturated group is an alkyne and the second reactiveunsaturated group is an azide. In certain other embodiments, the firstreactive unsaturated group is an azide and the second reactiveunsaturated group is an alkyne. In some embodiments, the presentinvention provides polypeptides labeled by the methods described herein.In some embodiments, the method of labeling a polypeptide takes place ina cell. In some embodiments, the method of labeling a polypeptide takesplace in a whole organism.

The present invention also provides methods of measuring proteinsynthesis in a cell or organism. For example, in certain embodiments,the present invention provides methods of measuring protein synthesis ina cell or organism comprising contacting a cell or organism with aneffective amount of a puromycin analog comprising a first reactivegroup, such that the puromycin analog is covalently bound to theC-terminus of one or more polypeptides in the cell to form one or morepolypeptide-puromycin analog conjugates; contacting the cell or organismwith a compound comprising a second reactive group and a label, suchthat a bioorthogonal reaction occurs between the first and secondreactive groups; and determining the amount of labeled protein in thecell to measure protein synthesis. In certain embodiments, the presentinvention provides methods of measuring protein synthesis in a cellcomprising contacting a cell with an effective amount of a puromycinanalog comprising a first reactive unsaturated group, such that thepuromycin analog is covalently bound to the C-terminus of one or morepolypeptides in the cell to form one or more polypeptide-puromycinanalog conjugates; contacting the cell with a compound comprising asecond reactive unsaturated group and a label, such that a [3+2]cycloaddition occurs between the first and second reactive unsaturatedgroups; and determining the amount of labeled protein in the cell tomeasure protein synthesis. In certain embodiments, the present inventionprovides methods of measuring protein synthesis in an organismcomprising administering to an organism an effective amount of apuromycin analog comprising a first reactive unsaturated group, suchthat the puromycin analog is covalently bound to the C-terminus of oneor more polypeptides in the organism to form one or morepolypeptide-puromycin analog conjugates; contacting at least one cell ofthe organism with a compound comprising a second reactive unsaturatedgroup and a label, such that a [3+2] cycloaddition occurs between thefirst and second reactive unsaturated groups; and determining the amountof labeled protein in the at least one cell in order to measure proteinsynthesis in the organism.

In another aspect, the present invention also provides screeningmethods. For example, in certain embodiments, the present inventionprovides a method of identifying an agent that perturbs proteinsynthesis comprising: contacting a cell or organism with a test agent;contacting the cell or organism with an effective amount of a puromycinanalog comprising a first reactive group, such that the puromycin analogis covalently bound to the C-terminus of nascent polypeptides in thecell; contacting the cell or organism with a compound comprising asecond reactive group and a label, such that a bioorthogonal reactionoccurs between the first and second reactive groups; determining theamount of label incorporated into nascent polypeptides, wherein theamount of label indicates the extent of protein synthesis; andidentifying the test agent as an agent that perturbs cellularproliferation if the amount of label incorporated into nascentpolypeptides is less than or greater than the amount of label measuredin a control in which a cell is not contacted with the test agent. Incertain embodiments, the present invention provides a method ofidentifying an agent that perturbs protein synthesis comprising:contacting a cell with a test agent; contacting the cell with aneffective amount of a puromycin analog comprising a first reactiveunsaturated group, such that the puromycin analog is covalently bound tothe C-terminus of nascent polypeptides in the cell; contacting the cellwith a compound comprising a second reactive unsaturated group and alabel, such that a [3+2] cycloaddition occurs between the first andsecond reactive unsaturated groups; determining the amount of labelincorporated into nascent polypeptides, wherein the amount of labelindicates the extent of protein synthesis; and identifying the testagent as an agent that perturbs cellular proliferation if the amount oflabel incorporated into nascent polypeptides is less than or greaterthan the amount of label measured in a control in which a cell is notcontacted with the test agent. In certain embodiments, the presentinvention provides methods of identifying an agent that perturbs proteinsynthesis in an organism comprising exposing an organism to a testagent; administering to the organism an effective amount of a puromycinanalog comprising a first reactive unsaturated group, such that thepuromycin analog is covalently bound to the C-terminus of nascentpolypeptides in the organism; contacting at least one cell of theorganism with a compound comprising a second reactive unsaturated groupand a label, such that a [3+2] cycloaddition occurs between the firstand second reactive unsaturated groups; determining the amount of labelincorporated into the nascent polypeptides in at least one cell, whereinthe amount of label indicates the extent of protein synthesis; andidentifying the test agent as an agent that perturbs cellularproliferation if the amount of label incorporated into nascentpolypeptides is less than or greater than the amount of label measuredin a control in which an organism is not administered the test agent.

The present invention also provides methods of isolating and/oridentifying nascent polypeptides. For example, in certain embodiments,the present invention provides a method of identifying a nascentpolypeptide comprising contacting a cell with an effective amount of apuromycin analog comprising a first reactive group, such that thepuromycin analog is covalently bound to the C-terminus of a nascentpolypeptide in the cell to form a polypeptide-puromycin analogconjugate; contacting the cell with a compound comprising a secondreactive group and an affinity label, such that a bioorthogonal reactionoccurs between the first and second reactive groups to form anaffinity-labeled polypeptide; and affinity purifying theaffinity-labeled polypeptide. In certain embodiments, the presentinvention provides a method of identifying a nascent polypeptidecomprising contacting a cell with an effective amount of a puromycinanalog comprising a first reactive unsaturated group, such that thepuromycin analog is covalently bound to the C-terminus of a nascentpolypeptide in the cell to form a polypeptide-puromycin analogconjugate; contacting the cell with a compound comprising a secondreactive unsaturated group and an affinity label, such that a [3+2]cycloaddition occurs between the first and second reactive unsaturatedgroups to form an affinity-labeled polypeptide; and affinity purifyingthe affinity-labeled polypeptide. In other embodiments, the presentinvention provides a method of identifying a nascent polypeptidecomprising contacting a cell with an effective amount of a puromycinanalog comprising a first reactive group, such that the puromycin analogis covalently bound to the C-terminus of one or more nascentpolypeptides in the cell to form one or more polypeptide-puromycinanalog conjugates; contacting the cell with a solid support comprising asecond reactive group, such that a bioorthogonal reaction occurs betweenthe first and second reactive groups; and identifying the nascentpolypeptide. In certain embodiments, the present invention provides amethod of identifying a nascent polypeptide comprising contacting a cellwith an effective amount of a puromycin analog comprising a firstreactive unsaturated group, such that the puromycin analog is covalentlybound to the C-terminus of one or more nascent polypeptides in the cellto form one or more polypeptide-puromycin analog conjugates; contactingthe cell with a solid support comprising a second reactive unsaturatedgroup, such that a [3+2] cycloaddition occurs between the first andsecond reactive unsaturated groups; and identifying the nascentpolypeptide. In certain embodiments, a method of identifying furthercomprises cleaving a polypeptide from a surface.

In another aspect, the invention provides a kit comprising a puromycinanalog of the present invention; and a compound comprising a label. Insome embodiments, the invention provides a kit comprising a puromycinanalog of the present invention comprising a first reactive unsaturatedgroup; and a compound comprising a second reactive unsaturated group anda label. In some embodiments, the kit further comprises Cu(I). In someembodiments, the kit further comprises an aqueous medium. In someembodiments, the kit further comprises instructions for use. These andother aspects of the invention will be described in further detail inconnection with the detailed description of the invention.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito: 1999, the entire contents of which are incorporatedherein by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex mixtures of isomers.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

The “enantiomeric excess” of a substance is a measure of how pure adesired enantiomer is relative to the undesired enantiomer. Enantiomericexcess is defined as the absolute difference between the mole fractionof each enantiomer which is most often expressed as a percentenantiomeric excess. For mixtures of diastereomers, there are analogousdefinitions and uses for “diastereomeric excess” and percentdiastereomeric excess. For example, a sample with 70% of R isomer and30% of S will have an enantiomeric excess of 40%. This can also bethought of as a mixture of 40% pure R with 60% of a racemic mixture(which contributes 30% R and 30% S to the overall composition).

The term “acyl,” as used herein, refers to a group having the generalformula —C(═O)R^(X1), —C(═O)OR^(X1), —C(═O)—O—C(═O)R^(X1),—C(═O)SR^(X1), —C(═O)N(R^(X1))₂, —C(═S)R^(X1), —C(═S)N(R^(X1))₂, and—C(═S)S(R^(X1)), —C(═NR^(X1))R^(X1), —C(═NR^(X1))OR^(X1),—C(═NR^(X1))SR^(X1), and —C(═NR^(X1))N(R^(X1))₂, wherein R^(X1) ishydrogen; halogen; substituted or unsubstituted hydroxyl; substituted orunsubstituted thiol; substituted or unsubstituted amino; substituted orunsubstituted acyl, cyclic or acyclic, substituted or unsubstituted,branched or unbranched aliphatic; cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic; cyclic oracyclic, substituted or unsubstituted, branched or unbranched alkyl;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedalkenyl; substituted or unsubstituted alkynyl; substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- ordi-aliphaticamino, mono- or di-heteroaliphaticamino, mono- ordi-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, ormono- or di-heteroarylamino; or two R^(X1) groups taken together form a5- to 6-membered heterocyclic ring. Exemplary acyl groups includealdehydes (—CHO), carboxylic acids (—CO₂H), ketones, acyl halides,esters, amides, imines, carbonates, carbamates, and ureas. Acylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “acyloxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted acyl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule.

The term “aliphatic,” as used herein, includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, and cyclic (i.e., carbocyclic) hydrocarbons, which areoptionally substituted with one or more functional groups. As will beappreciated by one of ordinary skill in the art, “aliphatic” is intendedherein to include, but is not limited to, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as usedherein, the term “alkyl” includes straight, branched and cyclic alkylgroups. An analogous convention applies to other generic terms such as“alkenyl”, “alkynyl”, and the like. Furthermore, as used herein, theterms “alkyl”, “alkenyl”, “alkynyl”, and the like encompass bothsubstituted and unsubstituted groups. In certain embodiments, as usedherein, “aliphatic” is used to indicate those aliphatic groups (cyclic,acyclic, substituted, unsubstituted, branched or unbranched) having 1-20carbon atoms. Aliphatic group substituents include, but are not limitedto, any of the substituents described herein, that result in theformation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

As used herein, the term “alkyl” is given its ordinary meaning in theart and refers to the radical of saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In some cases, the alkyl group may be a loweralkyl group, i.e., an alkyl group having 1 to 10 carbon atoms (e.g.,methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, ordecyl). In some embodiments, a straight chain or branched chain alkylmay have 30 or fewer carbon atoms in its backbone, and, in some cases,20 or fewer. In some embodiments, a straight chain or branched chainalkyl may have 12 or fewer carbon atoms in its backbone (e.g., C₁-C₁₂for straight chain, C₃-C₁₂ for branched chain), 6 or fewer, or 4 orfewer. Likewise, cycloalkyls may have from 3-10 carbon atoms in theirring structure, or 5, 6, or 7 carbons in the ring structure. Examples ofalkyl groups include, but are not limited to, methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, cyclobutyl, hexyl, andcyclochexyl.

The term “alkylene” as used herein refers to a bivalent alkyl group. An“alkylene” group is a polymethylene group, i.e., —(CH₂)_(z)—, wherein zis a positive integer, e.g., from 1 to 20, from 1 to 10, from 1 to 6,from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substitutedalkylene chain is a polymethylene group in which one or more methylenehydrogen atoms are replaced with a substituent. Suitable substituentsinclude those described herein for a substituted aliphatic group.

The terms “alkenyl” and “alkynyl” are given their ordinary meaning inthe art and refer to unsaturated aliphatic groups analogous in lengthand possible substitution to the alkyls described above, but thatcontain at least one double or triple bond respectively.

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-10 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-8 aliphatic carbon atoms. In still other embodiments, the alkyl,alkenyl, and alkynyl groups employed in the invention contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-4 carbon atoms.Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl,isobutyl, t-butyl, n-pentyl, sec-pentyl, isopentyl, t-pentyl, n-hexyl,sec-hexyl, moieties and the like, which again, may bear one or moresubstituents. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike. Representative alkynyl groups include, but are not limited to,ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.

The term “cycloalkyl,” as used herein, refers specifically to groupshaving three to ten, preferably three to seven carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other aliphatic, heteroaliphatic, or hetercyclic moieties, mayoptionally be substituted with substituents including, but not limitedto aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

The term “heteroaliphatic,” as used herein, refers to an aliphaticmoiety, as defined herein, which includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, cyclic (i.e., heterocyclic), or polycyclic hydrocarbons, whichare optionally substituted with one or more functional groups, and thatcontain one or more oxygen, sulfur, nitrogen, phosphorus, or siliconatoms, e.g., in place of carbon atoms. In certain embodiments,heteroaliphatic moieties are substituted by independent replacement ofone or more of the hydrogen atoms thereon with one or more substituents.As will be appreciated by one of ordinary skill in the art,“heteroaliphatic” is intended herein to include, but is not limited to,heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,heterocycloalkenyl, and heterocycloalkynyl moieties. Thus, the term“heteroaliphatic” includes the terms “heteroalkyl,” “heteroalkenyl”,“heteroalkynyl”, and the like. Furthermore, as used herein, the terms“heteroalkyl”, “heteroalkenyl”, “heteroalkynyl”, and the like encompassboth substituted and unsubstituted groups. In certain embodiments, asused herein, “heteroaliphatic” is used to indicate those heteroaliphaticgroups (cyclic, acyclic, substituted, unsubstituted, branched orunbranched) having 1-20 carbon atoms. Heteroaliphatic group substituentsinclude, but are not limited to, any of the substituents describedherein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano,isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, acyloxy, and the like, each of which may or may not befurther substituted).

The term “heteroalkyl” is given its ordinary meaning in the art andrefers to an alkyl group as described herein in which one or more carbonatoms is replaced by a heteroatom. Suitable heteroatoms include oxygen,sulfur, nitrogen, phosphorus, and the like. Examples of heteroalkylgroups include, but are not limited to, alkoxy, amino, thioester,poly(ethylene glycol), and alkyl-substituted amino.

The terms “heteroalkenyl” and “heteroalkynyl” are given their ordinarymeaning in the art and refer to unsaturated aliphatic groups analogousin length and possible substitution to the heteroalkyls described above,but that contain at least one double or triple bond respectively.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to, aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CHF₂; —CH₂F; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, alycyclic, heteroaliphatic, heterocyclic, aryl,heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic,heteroaliphatic, alkylaryl, or alkylheteroaryl substituents describedabove and herein may be substituted or unsubstituted, branched orunbranched, cyclic or acyclic, and wherein any of the aryl or heteroarylsubstituents described above and herein may be substituted orunsubstituted. Additional examples of generally applicable substituentsare illustrated by the specific embodiments shown in the Examples thatare described herein.

The term “aryl” is given its ordinary meaning in the art and refers toaromatic carbocyclic groups, optionally substituted, having a singlering (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fusedrings in which at least one is aromatic (e.g.,1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl). That is,at least one ring may have a conjugated pi electron system, while other,adjoining rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls. The aryl group may be optionally substituted, asdescribed herein. Substituents include, but are not limited to, any ofthe previously mentioned substitutents, i.e., the substituents recitedfor aliphatic moieties, or for other moieties as disclosed herein,resulting in the formation of a stable compound. In some cases, an arylgroup is a stable mono- or polycyclic unsaturated moiety havingpreferably 3-14 carbon atoms, each of which may be substituted orunsubstituted. “Carbocyclic aryl groups” refer to aryl groups whereinthe ring atoms on the aromatic ring are carbon atoms. Carbocyclic arylgroups include monocyclic carbocyclic aryl groups and polycyclic orfused compounds (e.g., two or more adjacent ring atoms are common to twoadjoining rings) such as naphthyl groups.

The terms “heteroaryl” is given its ordinary meaning in the art andrefers to aryl groups comprising at least one heteroatom as a ring atom.A “heteroaryl” is a stable heterocyclic or polyheterocyclic unsaturatedmoiety having preferably 3-14 carbon atoms, each of which may besubstituted or unsubstituted. Substituents include, but are not limitedto, any of the previously mentioned substitutents, i.e., thesubstituents recited for aliphatic moieties, or for other moieties asdisclosed herein, resulting in the formation of a stable compound. Insome cases, a heteroaryl is a cyclic aromatic radical having from fiveto ten ring atoms of which one ring atom is selected from S, O, and N;zero, one, or two ring atoms are additional heteroatoms independentlyselected from S, O, and N; and the remaining ring atoms are carbon, theradical being joined to the rest of the molecule via any of the ringatoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and thelike.

It will also be appreciated that aryl and heteroaryl moieties as definedherein may be attached via an alkyl or heteroalkyl moiety and thus alsoinclude -(alkyl)aryl, -(heteroalkyl)aryl, -(heteroalkyl)heteroaryl, and-(heteroalkyl)heteroaryl moieties. Thus, as used herein, the phrases“aryl or heteroaryl moieties” and “aryl, heteroaryl, -(alkyl)aryl,-(heteroalkyl)aryl, -(heteroalkyl)heteroaryl, and-(heteroalkyl)heteroaryl” are interchangeable. Substituents include, butare not limited to, any of the previously mentioned substituents, i.e.,the substituents recited for aliphatic moieties, or for other moietiesas disclosed herein, resulting in the formation of a stable compound.

It will be appreciated that aryl and heteroaryl groups (includingbicyclic aryl groups) can be unsubstituted or substituted, whereinsubstitution includes replacement of one or more of the hydrogen atomsthereon independently with any one or more of the following moietiesincluding, but not limited to: aliphatic; alicyclic; heteroaliphatic;heterocyclic; aromatic; heteroaromatic; aryl; heteroaryl; alkylaryl;heteroalkylaryl; alkylheteroaryl; heteroalkylheteroaryl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH; —NO₂; —CN; —CF₃;—CH₂F; —CHF₂; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃;—C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x);—OCON(R_(x))₂; —N(R_(x))₂; —S(O)R_(x); —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, alicyclic, heteroaliphatic, heterocyclic,aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl or heteroalkylheteroaryl, wherein any of the aliphatic,alicyclic, heteroaliphatic, heterocyclic, alkylaryl, or alkylheteroarylsubstituents described above and herein may be substituted orunsubstituted, branched or unbranched, saturated or unsaturated, andwherein any of the aromatic, heteroaromatic, aryl, heteroaryl,-(alkyl)aryl or -(alkyl)heteroaryl substituents described above andherein may be substituted or unsubstituted. Additionally, it will beappreciated, that any two adjacent groups taken together may represent a4, 5, 6, or 7-membered substituted or unsubstituted alicyclic orheterocyclic moiety. Additional examples of generally applicablesubstituents are illustrated by the specific embodiments describedherein.

The term “aryloxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted aryl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo,—Br), and iodine (iodo, —I).

The term “heterocycle” is given its ordinary meaning in the art andrefers to cyclic groups containing at least one heteroatom as a ringatom, in some cases, 1 to 3 heteroatoms as ring atoms, with theremainder of the ring atoms being carbon atoms. Suitable heteroatomsinclude oxygen, sulfur, nitrogen, phosphorus, and the like. In somecases, the heterocycle may be 3- to 10-membered ring structures or 3- to7-membered rings, whose ring structures include one to four heteroatoms.

The term “heterocycle” may include heteroaryl groups, saturatedheterocycles (e.g., cycloheteroalkyl) groups, or combinations thereof.The heterocycle may be a saturated molecule, or may comprise one or moredouble bonds. In some cases, the heterocycle is a nitrogen heterocycle,wherein at least one ring comprises at least one nitrogen ring atom. Theheterocycles may be fused to other rings to form a polycylicheterocycle. The heterocycle may also be fused to a spirocyclic group.In some cases, the heterocycle may be attached to a compound via anitrogen or a carbon atom in the ring.

Heterocycles include, for example, thiophene, benzothiophene,thianthrene, furan, tetrahydrofuran, pyran, isobenzofuran, chromene,xanthene, phenoxathiin, pyrrole, dihydropyrrole, pyrrolidine, imidazole,pyrazole, pyrazine, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,triazole, tetrazole, oxazole, isoxazole, thiazole, isothiazole,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, oxazine, piperidine, homopiperidine(hexamnethyleneimine), piperazine (e.g., N-methyl piperazine),morpholine, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, other saturated and/or unsaturated derivativesthereof, and the like. The heterocyclic ring can be optionallysubstituted at one or more positions with such substituents as describedherein. In some cases, the heterocycle may be bonded to a compound via aheteroatom ring atom (e.g., nitrogen). In some cases, the heterocyclemay be bonded to a compound via a carbon ring atom. In some cases, theheterocycle is pyridine, imidazole, pyrazine, pyrimidine, pyridazine,acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline,benzoquinoline, benzoisoquinoline, phenanthridine-1,9-diamine, or thelike.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “amino,” as used herein, refers to a primary (—NH₂), secondary(—NHR_(x)), tertiary (—NR_(x)R_(y)), or quaternary (—N⁺R_(x)R_(y)R_(z))amine, where R_(x), R_(y), and R_(z) are independently an aliphatic,alicyclic, heteroaliphatic, heterocyclic, aryl, or heteroaryl moiety, asdefined herein. Examples of amino groups include, but are not limitedto, methylamino, dimethylamino, ethylamino, diethylamino,methylethylamino, iso-propylamino, piperidino, trimethylamino, andpropylamino.

The term “alkyne” is given its ordinary meaning in the art and refers tobranched or unbranched unsaturated hydrocarbon groups containing atleast one triple bond. Non-limiting examples of alkynes includeacetylene, propyne, 1-butyne, 2-butyne, and the like. The alkyne groupmay be substituted and/or have one or more hydrogen atoms replaced witha functional group, such as a hydroxyl, halogen, alkoxy, and/or arylgroup.

The term “alkoxy” (or “alkyloxy”), or “thioalkyl” as used herein refersto an alkyl group, as previously defined, attached to the parentmolecular moiety through an oxygen atom or through a sulfur atom. Incertain embodiments, the alkyl group contains 1-20 aliphatic carbonatoms. In certain other embodiments, the alkyl group contains 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-8 aliphaticcarbon atoms. In still other embodiments, the alkyl group contains 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl groupcontains 1-4 aliphatic carbon atoms. Examples of alkoxy, include but arenot limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,t-butoxy, neopentoxy and n-hexoxy. Examples of thioalkyl include, butare not limited to, methylthio, ethylthio, propylthio, isopropylthio,n-butylthio, and the like.

The term “aryloxy” refers to the group, —O-aryl.

The term “alkoxyalkyl” refers to an alkyl group substituted with atleast one alkoxy group (e.g., one, two, three, or more, alkoxy groups).For example, an alkoxyalkyl group may be —(C₁₋₆-alkyl)-O—(C₁₋₆-alkyl),optionally substituted. In some cases, the alkoxyalkyl group may beoptionally substituted with another alkyoxyalkyl group (e.g.,—(C₁₋₆-alkyl)-O—(C₁₋₆-alkyl)-O—(C₁₋₆-alkyl), optionally substituted.

It will be appreciated that the above groups and/or compounds, asdescribed herein, may be optionally substituted with any number ofsubstituents or functional moieties. That is, any of the above groupsmay be optionally substituted. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds, “permissible” being in the context of the chemical rules ofvalence known to those of ordinary skill in the art. In general, theterm “substituted” whether preceeded by the term “optionally” or not,and substituents contained in formulas of this invention, refer to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. When more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. It will be understood that “substituted”also includes that the substitution results in a stable compound, e.g.,which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc. In some cases,“substituted” may generally refer to replacement of a hydrogen with asubstituent as described herein. However, “substituted,” as used herein,does not encompass replacement and/or alteration of a key functionalgroup by which a molecule is identified, e.g., such that the“substituted” functional group becomes, through substitution, adifferent functional group. For example, a “substituted phenyl group”must still comprise the phenyl moiety and cannot be modified bysubstitution, in this definition, to become, e.g., a pyridine ring. In abroad aspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnonaromatic substituents of organic compounds. Illustrative substituentsinclude, for example, those described herein. The permissiblesubstituents can be one or more and the same or different forappropriate organic compounds. For purposes of this invention, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valencies of the heteroatoms. Furthermore, this invention isnot intended to be limited in any manner by the permissible substituentsof organic compounds. Combinations of substituents and variablesenvisioned by this invention are preferably those that result in theformation of stable compounds useful for the formation of an imagingagent or an imaging agent precursor. The term “stable,” as used herein,preferably refers to compounds which possess stability sufficient toallow manufacture and which maintain the integrity of the compound for asufficient period of time to be detected and preferably for a sufficientperiod of time to be useful for the purposes detailed herein.

Examples of substituents include, but are not limited to, halogen,azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromaticmoieties, —CF₃, —CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl,heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, amino, halide,alkylthio, oxo, acylalkyl, carboxy esters, -carboxamido, acyloxy,aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl,arylamino, aralkylamino, alkylsulfonyl, -carboxamidoalkylaryl,-carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy-,aminocarboxamidoalkyl-, cyano, alkoxyalkyl, perhaloalkyl,arylalkyloxyalkyl, and the like.

In certain embodiments, the substituent present on the nitrogen atom isan amino protecting group (also referred to herein as a “nitrogenprotecting group”). Amino protecting groups include, but are not limitedto, —OH, —OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂,—CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc) and R^(dd) are as defined herein. Amino protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

For example, amino protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Amino protecting groups such as carbamate groups (e.g., —C(═O)OR^(aa))include, but are not limited to, methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Amino protecting groups such as sulfonamide groups (e.g., —S(═O)₂R^(aa))include, but are not limited to, p-toluenesulfonamide (Ts),benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other amino protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is ahydroxyl protecting group (also referred to herein as an “oxygenprotecting group”). Hydroxyl protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Hydroxyl protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary hydroxyl protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

The term “independently selected” is used herein to indicate that the Rgroups can be identical or different.

“Polypeptide,” “peptide,” or “protein”: According to the presentinvention, a “polypeptide,” “peptide,” “protein” comprises a string ofat least two amino acids linked together by peptide bonds. The terms“protein,” “peptide,” or “polypeptide” may be used interchangeably.Peptide may refer to an individual peptide or a collection ofpolypeptides. In certain embodiments, inventive peptides contain onlynatural amino acids, although non-natural amino acids (i.e., compoundsthat do not occur in nature but that can be incorporated into apolypeptide chain) and/or amino acid analogs as are known in the art mayalternatively be employed. Also, one or more of the amino acids in aninventive peptide may be modified, for example, by the addition of achemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification. In certainembodiments, the modifications of the peptide lead to a more stablepeptide (e.g., greater half-life in vivo). These modifications mayinclude cyclization of the peptide, the incorporation of D-amino acids,etc. None of the modifications should substantially interfere with thedesired biological activity of the peptide. In some embodiments, apolypeptide according to the present invention is conjugated to apuromycin analog. In some embodiments, a polypeptide according to thepresent invention is conjugated to a puromycin analog that is attachedto a detectable label.

As used herein, the term “reactive group” refers to a functional groupcapable of undergoing a bioorthogonal reaction. A “bioorthogonalreaction,” as used herein, refers to a reaction that can be performed ina biological system without interfering with biological processes. Abioorthogonal reaction generally has a fast rate under physiologicalconditions and is inert to chemical functionalities found in vivo. Someexamples of bioorthogonal reactions include, but are not limited to,[3+2] cycloadditions, Staudinger ligation, oxime ligation or hydrazoneligation (Dirksen et al., Biocong. Chem. 19:2543-2548 (2008)), inverseelectron demand Diels-Alder (e.g., tetrazine ligation (Blackman et al.,J. Am. Chem. Soc. 130:13518-13519 (2008))), and [2+2+2] cycloaddition(e.g., quadricyclane ligation (Sletten et al., J. Am. Chem. Soc.133:17570-17573 (2011))).

As used herein, the term “reactive unsaturated group” refers to afunctional group containing atoms sharing more than one valence bond andthat can undergo addition reactions, in particular cycloadditions. Areactive unsaturated group typically possesses at least one double ortriple bond. In addition to the reactive moiety itself (e.g., double ortriple bonded atoms), a reactive unsaturated group optionally comprisesan alkyl or heteroalkyl linker moiety of 1-6 atoms.

The term “1,3-dipole” has herein its art understood meaning and refersto a molecule or functional group that is isoelectronic with the allylanion and has four electrons in a π system encompassing the 1,3-dipole.1,3-Dipoles generally have one or more resonance structures showing thecharacteristic 1,3-dipole. Examples of 1,3-dipoles include nitrileoxides, azides, diazomethanes, nitrones, and nitrile imines.

As used herein, the term “dipolarophile” has its art understood meaningand refers to a molecule or functional group that contains a π bond andthat exhibits reactivity toward 1,3-dipoles. The reactivity ofdipolarophiles depends both on the substituents present on the π bondand on the nature of the 1,3-dipole involved in the reaction.Dipolarophiles are typically alkenes or alkynes.

As used herein, the term “cycloaddition” refers to a chemical reactionin which two or more π-electron systems (e.g., unsaturated molecules orunsaturated parts of the same molecule) combine to form a cyclic productin which there is a net reduction of the bond multiplicity. In acycloaddition, the π electrons are used to form new a bonds. The productof a cycloaddition is called an “adduct” or “cycloadduct”. Differenttypes of cycloadditions are known in the art including, but not limitedto, [3+2] cycloadditions and Diels-Alder reactions. [3+2]Cycloadditions, which are also called 1,3-dipolar cycloadditions, occurbetween a 1,3-dipole and a dipolarophile and are typically used for theconstruction of five-membered heterocyclic rings. The term “[3+2]cycloaddition” also encompasses “copperless” [3+2] cycloadditionsbetween azides and cyclooctynes and difluorocyclooctynes described byBertozzi et al., J. Am. Chem. Soc., 2004, 126: 15046-15047).

The terms “labeled”, “labeled with a detectable agent”, and “labeledwith a detectable moiety” are used herein interchangeably. “Label” and“detectable moiety” are also used interchangeably herein. When used inreference to a polypeptide, these terms specify that the polypeptide canbe detected or visualized. In certain embodiments, a label is selectedsuch that it generates a signal which can be measured and whoseintensity is related to the amount of labeled polypeptides (e.g., in asample). A label may be directly detectable (i.e., it does not requireany further reaction or manipulation to be detectable, e.g., afluorophore is directly detectable) or it may be indirectly detectable(i.e., it is made detectable through reaction or binding with anotherentity that is detectable, e.g., a hapten is detectable byimmunostaining after reaction with an appropriate antibody comprising areporter such as a fluorophore). Labels suitable for use in the presentinvention may be detectable by any of a variety of means including, butnot limited to, spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Suitable labelsinclude, but are not limited to, various ligands, radionuclides,fluorescent dyes, chemiluminescent agents, microparticles, enzymes,calorimetric labels, magnetic labels, and haptens.

The terms “fluorophore”, “fluorescent moiety” and “fluorescent dye” areused herein interchangeably. They refer to a molecule which, in solutionand upon excitation with light of appropriate wavelength, emits lightback, generally at a longer wavelength. Numerous fluorescent dyes of awide variety of structures and characteristics are suitable for use inthe practice of the present invention. In choosing a fluorophore, it isoften desirable that the molecule absorbs light and emits fluorescencewith high efficiency (i.e., the fluorescent molecule has a high molarextinction coefficient at the excitation wavelength and a highfluorescence quantum yield, respectively) and is photostable (i.e., thefluorescent molecule does not undergo significant degradation upon lightexcitation within the time necessary to perform the detection).

As used herein, the term “effective amount” refers to the amount of asubstance, compound, molecule, agent or composition that elicits therelevant response in a cell, a tissue, or an organism. For example, inthe case of a puromycin analog administered to an organism, an effectiveamount of puromycin analog is an amount of puromycin analog that isconjugated to nascent polypeptides in one or more cell of the organism.

As used herein, the term “organism” refers to a living system that hasor can develop the ability to act or function independently. An organismmay be unicellular or multicellular. Organisms include humans, animals,plants, bacteria, protozoa, and fungi.

As used herein, the term “puromycin analog” refers to a compound havingthe general aminonucleoside core structure of puromycin that is capableof conjugating to the C-terminus of a polypeptide and is modified toinclude a reactive group capable of undergoing a bioorthogonal reaction.In certain embodiments, “puromycin analog” refers to a compound havingthe general aminonucleoside core structure of puromycin that is capableof conjugating to the C-terminus of a polypeptide and is modified toinclude a reactive unsaturated group, such as a 1,3-dipole or adipolarophile. In some embodiments, a puromycin analog according to thepresent invention is of Formula (I) as described herein.

As used herein, the term “polypeptide-puromycin analog conjugate” refersto a polypeptide where a puromycin analog as described herein iscovalently conjugated to the C-terminus.

The terms “detectable polypeptide” and “labeled polypeptide” are usedinterchangeably herein, and refer to a polypeptide-puromycin analogconjugate that has undergone a bioorthogonal reaction and is therebyattached to a label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that O-propargyl-puromycin (OP-puro), an alkyne puromycin(puro) analog, is a potent protein synthesis inhibitor. (A) Structure ofpuro and the analog OP-puro, which bears a terminal alkyne group. (B)Schematic of OP-puro incorporation into nascent polypeptide chains ontranslating ribosomes. The prematurely terminated polypeptides aresubsequently detected by copper(I)-catalyzed azide-alkyne cycloaddition(CuAAC) using a fluorescent azide. (C) Inhibition of protein translationin vitro by puro and OP-puro. A ³⁵S-methionine-labeled protein (a GFPfusion of mouse Suppressor of Fused) was generated by translation inrabbit reticulocyte lysates, in the absence or presence of varyingconcentrations of puro and OP-puro. The translation reactions wereseparated by SDS-PAGE, and the translated protein was visualized byautoradiography. OP-puro inhibits protein synthesis in a dose-dependentmanner. (D) Inhibition of protein translation in cultured cells by puroand OP-puro. Human embryonic kidney 293T cells were incubated inmethionine-free media supplemented with ³⁵S-methionine, in the absenceor presence of varying concentrations of puro and OP-puro. Total celllysates were analyzed by SDS-PAGE, followed by autoradiography, tomeasure bulk protein translation. The gel was also stained withCoomassie Blue, to demonstrate equal protein loading. (E) Formation ofconjugates between OP-puro and nascent polypeptide chains. Cultured 293Tcells were labeled with ³⁵S-methionine as in D), in the presence ofOP-puro, puro or OP-puro and the protein synthesis inhibitor,cycloheximide (CHX). Cellular lysates were reacted with biotin-azideunder conditions for CuAAC, after which biotinylated molecules werepurified on streptavidin beads. Bound proteins were eluted, separated bySDS-PAGE, followed by autoradiography to detect nascent proteins.

FIG. 2 depicts imaging of nascent proteins in cultured cells withOP-puro. (A) Cultured NIH-3T3 cells were incubated for 1 hour incomplete media supplemented with increasing concentrations of OP-puro,OP-puro and cycloheximide (CHX), or control vehicle. The cells were thenfixed, stained by CuAAC with Alexa568-azide and imaged by fluorescencemicroscopy. A specific signal is observed in cells treated with OP-puro,which is proportional to the concentration of added OP-puro; this signalis abolished if protein translation is blocked with CHX (50micrograms/mL), which dissociates ribosomes and thus prevents theformation of conjugates between nascent polypeptide chains and OP-puro.(B) Time course of OP-puro incorporation into nascent proteins. NIH-3T3cells were incubated with OP-puro (50 microM, which is sufficient tocompletely block protein synthesis) for 14 varying amounts of time,after which OP-puro incorporation was imaged as in (A). The intensity ofthe OP-puro signal reaches a maximum after about 1 hour. (C) The nascentprotein-OP-puro conjugates are unstable and are cleared from cells in aproteasome-dependent manner. NIH-3T3 cells were treated with 50 microMOP-puro for 15 minutes, followed by incubation in media without OP-puro,in the absence or presence of 5 microM of the proteasome inhibitorbortezomib. Parallel cultures were fixed at the indicated times afterremoval of OP-puro, and nascent protein-OP-puro conjugates were imagedby CuAAC labelling using Alexa568-azide. The OP-puro conjugates havelargely disappeared after 1 hour but are completely stabilized byproteasome inhibition. Untreated cells and cells incubated for 15minutes with OP-puro (50 microM) and CHX (50 micrograms/mL) served asnegative controls.

FIG. 3 depicts the use of OP-puro to image protein synthesis in wholeanimals. One hundred microliters of a 20 mM OP-puro solution in PBS orPBS alone (negative control) were injected intraperitoneally into mice.Organs were harvested 1 hour later, fixed in formalin, and stained usingCuAAC with tetramethylrhodamine (TMR)-azide, either after paraffinsectioning or whole mount. (A) Section through mouse small intestineshowing intestinal vili sectioned longitudinally. OP-puro stainsstrongly the cells in the crypts (particularly Paneth cells) and thecells at the base of the villi. Bottom panels show a highermagnification (40× objective) view of the intestinal crypts in anOP-puro-injected mouse. Note the intense staining of the secretorygranules characteristic of Paneth cells. (B) Whole mount staining ofmouse small intestine, showing the localization of the OP-puro stain inthe crypts. Protein-OP-puro conjugates were detected with TMR-azide(red), while nuclear DNA was stained with OliGreen (green). (C) OP-puroincorporation into striated muscle fibers. Paraffin sections of musclewere stained as in (A). Sarcomeres are strongly stained with OP-puro,likely because some protein-OPpuro conjugates are functional and areproperly assembled into sarcomeres. Images of OP-puro staining of othersurveyed mouse tissues (spleen, kidney, liver) are shown in FIG. 4.

FIG. 4 depicts imaging of protein synthesis in whole animals withOP-puro. One hundred microliters of a 20 mM OP-puro solution in PBS orPBS alone (negative control) were injected intraperitoneally into mice.Organs were harvested 1 hour later and were fixed in formalin. Organfragments were embedded in paraffin, sectioned, and stained using CuAACwith 15 tetramethylrhodamine (TMR)-azide, followed by imaging byfluorescence microscopy and by DIC. (A) Section through mouse liver.OP-puro stains strongly all hepatocytes. (B) Section through mousekidney. (C) Section through mouse spleen.

FIG. 5 shows that O-azidoethyl-puromycin (AE-puro), an azido puromycin(puro) analog, is a potent protein synthesis inhibitor. (A) Structure ofAE-puro, which bears a terminal azide group. (B) Inhibition of proteintranslation in vitro by puro and AE-puro. A ³⁵S-methionine-labeledprotein (a GFP fusion of mouse Suppressor of Fused) was generated bytranslation in rabbit reticulocyte lysates, in the absence or presenceof varying concentrations of puro and AE-puro. The translation reactionswere separated by SDS-PAGE, and the translated protein was visualized byautoradiography. AE-puro inhibits protein synthesis in a dose-dependentmanner.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides methods and compositions for labelingpolypeptides and for measuring protein synthesis and identifying nascentpolypeptides both in vitro and in vivo.

Labeling of Polypeptides

In some embodiments, labeling methods of the present invention generallyinclude a bioorthogonal reaction between a first reactive group on apuromycin analog incorporated into a polypeptide and a second reactivegroup attached to a label. In some embodiments, labeling methods of thepresent invention generally include a [3+2] cycloaddition between afirst reactive unsaturated group on a puromycin analog incorporated intoa polypeptide and a second reactive unsaturated group attached to alabel. An example of such a labeling method is schematically presentedin FIG. 1B.

In certain embodiments, the present invention provides a method oflabeling a polypeptide comprising providing a polypeptide-puromycinanalog conjugate comprising a first reactive group; and contacting thepolypeptide-puromycin analog conjugate with a compound comprising asecond reactive group and a label, such that a bioorthogonal reactionoccurs between the first and second reactive groups. In certainembodiments, the present invention provides a method of labeling apolypeptide comprising providing a polypeptide-puromycin analogconjugate comprising a first reactive unsaturated group; and contactingthe polypeptide-puromycin analog conjugate with a compound comprising asecond reactive unsaturated group and a label, such that a [3+2]cycloaddition occurs between the first and second unsaturated groups.

Puromycin Analogs

Puromycin (puro) (FIG. 1A) is an aminonucleoside antibiotic that blocksprotein synthesis in both prokaryotes and eukaryotes, by causingpremature termination of nascent polypeptide chains. Puromycin mimics anaminoacyl-tRNA molecule and binds to the acceptor site of translatingribosomes, which leads to the formation of an amide bond between theC-terminus of the nascent polypeptide chain and the primary amine groupof puromycin (Nathans, Proc. Natl. Acad. Sci. USA 51:585-592 (1964);Nathans, Fed. Proc. 23:984-989 (1964)). The polypeptide chain-puroconjugate is then released from the ribosome, followed by its quick,ubiquitin-dependent proteolysis (Goldberg, Proc. Natl. Acad. Sci. USA69(2):422-426 (1972); Wharton et al., FEBS Lett. 168(1):134-138 (1984)).The translation inhibition mechanism of puro has been exploited in thepast to assay the rate of synthesis of specific proteins, using labelingwith radioactive puro followed by immunoprecipitation with antibodiesagainst the protein of interest (Issacs et al., Proc. Natl. Acad. Sci.USA 84(17):6174-6178 (1987)).

Puromycin analogs suitable for use in the practice of the methods of thepresent invention include any puromycin analog that contains a reactivegroup that can undergo a bioorthogonal reaction. In some embodiments,puromycin analogs suitable for use in the practice of the methods of thepresent invention include any puromycin analog that contains a reactiveunsaturated group that can undergo a [3+2] cycloaddition. In otherembodiments, puromycin analogs suitable for use in the practice of themethods of the present invention include any puromycin analog thatcontains a reactive group that can undergo a Staudinger ligation. Insome embodiments, puromycin analogs suitable for use in the practice ofthe methods of the present invention include any puromycin analog thatcontains a reactive group that can undergo an inverse electron demandDiels-Alder reaction (e.g., tetrazine ligation), oxime addition,hydrazone addition, or [2+2+2] cycloaddition (e.g., quadricyclaneligation). In some embodiments, the reactive unsaturated group iscarried by the amino acid side chain portion of the puromycin analog. Anamino acid side chain can be any side chain of a natural or non-naturalamino acid. For example, the side chain of phenylalanine is benzyl, theside chain of alanine is methyl, and the side chain of tyrosine is4-hydroxybenzyl.

In certain embodiments, a reactive unsaturated group is a 1,3-dipolesuch as a nitrile oxide, an azide, a diazomethane, a nitrone, or anitrile imine. In certain embodiments, the 1,3-dipole is an azide.Alternatively, a reactive unsaturated group can be a dipolarophile suchas an alkene (e.g., vinyl, propylenyl, and the like) or an alkyne (e.g.,ethynyl, propynyl, and the like). In certain embodiments, thedipolarophile is an alkyne, such as, for example, an ethynyl group or apropargyl group.

Methods for the preparation of puromycin analogs are known in the art.For example, see Pestka et al., Antimicrob Agents Chemother 4(1):37-43(1973); Eckermann et al., Eur J Biochem 41(3):547-554 (1974); Vanin etal., FEBS Lett 40(1):124-126 (1974); Lee et al., J Med Chem24(3):304-308 (1981).

In certain embodiments, a puromycin analog of the present invention is acompound of Formula (I):

wherein

R^(A) is a bond, or an optionally substituted group selected fromaliphatic, heteroaliphatic, aryl, and heteroaryl, or a combinationthereof;

R^(B) is a bond or C₁₋₆ aliphatic;

R¹ is hydrogen or a reactive group capable of undergoing a bioorthogonalreaction;

R^(1′) is hydrogen or a reactive group capable of undergoing abioorthogonal reaction;

wherein R¹ and R^(1′) are not simultaneously hydrogen;

R² is hydrogen or C₁₋₆ aliphatic;

R³, R⁴, and R⁵ are each independently hydrogen or a protecting group;

R⁶ is hydrogen or C₁₋₆ aliphatic;

or a salt thereof.

In certain embodiments, a puromycin analog of the present invention is acompound of Formula (II):

wherein

R^(A) is a bond, or an optionally substituted group selected fromaliphatic, heteroaliphatic, aryl, and heteroaryl, or a combinationthereof;

R¹ is a reactive group capable of undergoing a bioorthogonal reaction;

R² is hydrogen or C₁₋₆ aliphatic;

R³, R⁴, and R⁵ are each independently hydrogen or a protecting group;

R⁶ is hydrogen or C₁₋₆ aliphatic;

or a salt thereof.

In certain embodiments, a puromycin analog of the present invention is acompound of Formula (III):

wherein

R^(A) is a bond, or an optionally substituted group selected fromaliphatic, heteroaliphatic, aryl, and heteroaryl, or a combinationthereof;

R¹ is a reactive unsaturated group;

R² is hydrogen or C₁₋₆ aliphatic;

R³, R⁴, and R⁵ are each independently hydrogen or a protecting group;

or a salt thereof.

In certain embodiments, a puromycin analog of the present invention is acompound of Formula (I-a), (II-a), or (III-a):

As defined generally above, R^(A) is a bond, or an optionallysubstituted group selected from aliphatic, heteroaliphatic, aryl, andheteroaryl, or a combination thereof. In some embodiments, R^(A) is abond. It will be understood by one of ordinary skill in the art thatwhen R^(A) is a bond, R¹ is directly attached to the α-carbon of themolecule, geminal to R². In some embodiments, R^(A) is a substitutedaliphatic group. In other embodiments, R^(A) is an unsubstitutedaliphatic group. In some embodiments, R^(A) is a substitutedheteroaliphatic group. In other embodiments, R^(A) is an unsubstitutedheteroaliphatic group. In some embodiments, R^(A) is a substituted arylgroup. In other embodiments, R^(A) is an unsubstituted aryl group. Insome embodiments, R^(A) is a substituted heteroaryl group. In otherembodiments, R^(A) is an unsubstituted heteroaryl group. In someembodiments, R^(A) is a side chain of a naturally occurring amino acid.In some embodiments, R^(A) is an aryl group. In some embodiments, R^(A)is a combination of aliphatic, heteroaliphatic, aryl, and/or heteroaryl.For example, in some embodiments, R^(A) is an -alkylaryl group. Incertain embodiments, R^(A) is a benzyl group. In some embodiments, R^(A)is a tyrosine side chain. In some embodiments, R^(A) is an alkyl group.In some embodiments, R^(A) is a C₁₋₃ alkyl group. In certainembodiments, R^(A) is C₁ or C₂ alkyl group. In some embodiments, R^(A)is a heteroaryl group. In some embodiments, R^(A) is a 5-6 memberedheteroaryl having 1-3 heteroatoms selected from nitrogen, oxygen, andsulfur. In certain embodiments, R^(A) is a 6-membered heteroaryl having1-3 nitrogens. In certain embodiments, R^(A) is a pyridyl group. Incertain embodiments, —R^(A)—R¹ is selected from:

As defined generally herein, R¹ is hydrogen or a reactive group capableof undergoing a bioorthogonal reaction. In some embodiments, R¹ ishydrogen. In certain embodiments, R¹ is a reactive unsaturated groupcapable of undergoing a [3+2] cycloaddition (sometimes referred to as“click” chemistry). In certain embodiments, R¹ comprises an azidecapable of undergoing a Staudinger ligation. In certain embodiments, R¹comprises an aldehyde. In certain embodiments, R¹ comprises a tetrazine.In certain embodiments, R¹ comprises a trans-cyclooctene.

In certain embodiments, R¹ is a reactive unsaturated group. As definedgenerally herein, a reactive unsaturated group is a functional groupcontaining atoms sharing more than one valence bond and that can undergoaddition reactions, in particular cycloadditions. In addition to thereactive moiety itself (e.g., double or triple bonded atoms), a reactiveunsaturated group optionally comprises an alkyl or heteroalkyl linkermoiety of 1-6 atoms. For example, in some embodiments, a reactiveunsaturated group is a group selected from propargyloxy, ethynyl,propargyl, homopropargyl, azidoethoxy, azido, azidomethyl, orazidoethyl. In some embodiments, R¹ is a reactive unsaturated group thatpossesses at least one double or triple bond. In some embodiments, R¹ isa reactive unsaturated group comprises a dipolarophile. In certainembodiments, R¹ is a reactive unsaturated group that comprises a triplebond. In certain embodiments, R¹ comprises an alkyne. In certain otherembodiments, R¹ comprises an alkene. In certain embodiments, R¹comprises an ethynyl group. In certain embodiments, R¹ is an ethynyl orpropargyl group. In other embodiments, R¹ is a reactive unsaturatedgroup that comprises a 1,3-dipole. In certain embodiments, R¹ is areactive unsaturated group that comprises a nitrile oxide, azide,diazomethane, nitrone, or nitrile imine. In certain embodiments, R¹comprises an azide. In certain embodiments, R¹ is an azide. In certainembodiments, R¹ is a azidoalkyl group, wherein the alkyl portion isC₁₋₆. In certain embodiments, R¹ is an azidomethyl group. In certainembodiments, R¹ is an azidoethyl group.

In some embodiments, an —R^(A)—R¹ group is selected from the groupconsisting of:

As defined generally above, R^(B) is a bond or C₁₋₆ aliphatic. In someembodiments, R^(B) is a bond. In some embodiments, R^(B) is C₁₋₆aliphatic. In some embodiments, R^(B) is a C₁₋₃ alkylene. In certainembodiments, R^(B) is C₁ or C₂ alkylene. In certain embodiments, R^(B)is methylene.

As defined generally herein, R^(1′) is hydrogen or a reactive groupcapable of undergoing a bioorthogonal reaction. In some embodiments,R^(1′) is hydrogen. In certain embodiments, R^(1′) is a reactiveunsaturated group capable of undergoing a [3+2] cycloaddition (sometimesreferred to as “click” chemistry). In certain embodiments, R^(1′)comprises an azide capable of undergoing a Staudinger ligation. Incertain embodiments, R^(1′) comprises an aldehyde. In certainembodiments, R^(1′) comprises a tetrazine. In certain embodiments,R^(1′) comprises a trans-cyclooctene.

In certain embodiments, R^(1′) is a reactive unsaturated group. Asdefined generally herein, a reactive unsaturated group is a functionalgroup containing atoms sharing more than one valence bond and that canundergo addition reactions, in particular cycloadditions. In addition tothe reactive moiety itself (e.g., double or triple bonded atoms), areactive unsaturated group optionally comprises an alkyl or heteroalkyllinker moiety of 1-6 atoms. For example, in some embodiments, a reactiveunsaturated group is a group selected from propargyloxy, ethynyl,propargyl, homopropargyl, azidoethoxy, azido, azidomethyl, orazidoethyl. In some embodiments, R^(1′) is a reactive unsaturated groupthat possesses at least one double or triple bond. In some embodiments,R^(1′) is a reactive unsaturated group comprises a dipolarophile. Incertain embodiments, R^(1′) is a reactive unsaturated group thatcomprises a triple bond. In certain embodiments, R^(1′) comprises analkyne. In certain other embodiments, R^(1′) comprises an alkene. Incertain embodiments, R¹ comprises an ethynyl group. In certainembodiments, R¹ is an ethynyl or propargyl group. In other embodiments,R^(1′) is a reactive unsaturated group that comprises a 1,3-dipole. Incertain embodiments, R^(1′) is a reactive unsaturated group thatcomprises a nitrile oxide, azide, diazomethane, nitrone, or nitrileimine. In certain embodiments, R^(1′) comprises an azide. In certainembodiments, R^(1′) is an azide. In certain embodiments, R^(1′) is aazidoalkyl group, wherein the alkyl portion is C₁₋₆. In certainembodiments, R^(1′) is an azidomethyl group. In certain embodiments,R^(1′) is an azidoethyl group. In certain embodiments, —R^(B)—R^(1′) ispropargyl.

In some embodiments, R¹ is hydrogen and R^(1′) is a reactive group. Insome embodiments, R¹ is a reactive group and R^(1′) is hydrogen. In someembodiments, R¹ is hydrogen and R^(1′) is a reactive unsaturated groupas defined herein. In some embodiments, R¹ is a reactive unsaturatedgroup as defined herein and R^(1′) is hydrogen.

In some embodiments, R¹ and R^(1′) are each reactive groups. In someembodiments, R¹ and R^(1′) are each reactive unsaturated groups. In someembodiments, R¹ and R^(1′) are orthogonal to one another. In someembodiments, R¹ and R^(1′) are the same. In some embodiments, R¹ andR^(1′) are different.

As defined generally above, R² is hydrogen or C₁₋₆ aliphatic. In certainembodiments, R² is hydrogen. In other embodiments, R² is C₁₋₆ aliphatic.In certain embodiments, R² is C₁₋₆ alkyl. In certain embodiments, R² isC₁₋₃ alkyl. In certain embodiments, R² is methyl or ethyl.

As defined generally above, R³, R⁴, and R⁵ are each independentlyhydrogen or a protecting group. In certain embodiments, R³, R⁴, and R⁵are each hydrogen. In other embodiments, R³, R⁴, and R⁵ are eachprotecting groups. In certain embodiments, at least one of R³, R⁴, andR⁵ is a protecting group. In certain embodiments, at least two of R³,R⁴, and R⁵ are protecting groups. In certain embodiments, R³ and R⁴ areprotecting groups, and R⁵ is hydrogen. In certain embodiments, R³ and R⁴are hydrogen, and R⁵ is a protecting group.

In certain embodiments, a puromycin analog according to the presentinvention is:

In certain embodiments, a puromycin analog according to the presentinvention is OP-puro or AE-puro.

Polypeptides and Polypeptide-Puromycin Analog Conjugates

In some embodiments, polypeptides are produced according to methods ofthe present invention or utilized in methods of the present invention.As will be appreciated by one of ordinary skill in the art, thepolypeptides can be of any of a wide range of lengths including shortpeptides comprising at least 2, 4, 6, 8, 10, 12, 15, 20, 25, 30, or 40amino acids as well as longer polypeptides and full length proteins.

In some embodiments, puromycin analogs can be incorporated into theproteome of a cell or organism. Isolation or purification of thepolypeptides and polypeptide-puromycin analog conjugates of the presentinvention, where necessary, may be carried out by any of a variety ofmethods well-known in the art. In some embodiments, purification ofpolypeptides and polypeptide-puromycin analog conjugates is performed byaffinity chromatography or reverse phase HPLC. In certain embodiments, apolypeptide-puromycin analog is purified by affinity chromatography viaan affinity label attached to the puromycin analog.

If desired, the sequence of polypeptides or polypeptide-puromycin analogconjugates can be verified using any suitable sequencing methodincluding, but not limited to, Edman degradation, matrix-assisted laserdesorption ionization time-of-flight (MALDI-TOF) mass spectrometry,liquid chromatography-mass spectrometry, and the like.

[3+2] Cycloaddition

In certain embodiments, methods provided herein generally include a[3+2] cycloaddition. In such methods, a [3+2] cycloaddition occursbetween a first reactive unsaturated group on a polypeptide-puromycinanalog conjugate and a second reactive unsaturated group on a reagentcomprising a label (also called herein a labeling reagent).

In some embodiments, a labeling reagent is selected such that the secondreactive unsaturated group can react via a [3+2] cycloaddition with thefirst reactive unsaturated group on the puromycin analog. Morespecifically, in certain embodiments, when the first unsaturated groupis a 1,3-dipole, the second unsaturated group will be a dipolarophilethat can react with the 1,3-dipole. In other embodiments, when the firstunsaturated group is a dipolarophile, the second unsaturated group willbe a 1,3-dipole that can react with the dipolarophile.

Optimization of [3+2] cycloaddition reaction conditions is within theskill of the art. In certain embodiments, the [3+2] cycloaddition isperformed under aqueous conditions.

In embodiments where the 1,3-dipole is an azide, and the dipolarophileis an alkyne (e.g., ethynyl group), the [3+2] cycloaddition may beperformed as described by Sharpless and coworkers (Rostovtsev et al.,Angew Chem. Int. Ed. Engl. 41:1596-1599 (2002); Lewis et al., AngewChem. Int. Ed. Engl. 41:1053-1057 (2002); Wang et al., J. Am. Chem. Soc.125:3192-3193 (2003)) at physiological temperatures, under aqueousconditions and in the presence of copper(I) (or Cu(I)), which catalyzesthe cycloaddition. This catalyzed version of the [3+2] cycloaddition istermed “click” chemistry.

In other embodiments, for example where the presence of exogenous Cu(I)is not desired (e.g., when Cu(I) is toxic to a living system), the [3+2]cycloaddition between the azide and the alkyne may be performed asdescribed by Sharpless and coworkers except for the presence of Cu(I).In certain embodiments, a labeling reagent used in a cycloadditioncomprises a copper chelating moiety in addition to a reactiveunsaturated group and a label. As used herein, the term “Cu chelatingmoiety” refers to any entity characterized by the presence of two ormore polar groups that can participate in the formation of a complex(containing more than one coordinate bond) with copper(I) ions. A copperchelating moiety can mobilize copper(I) ions naturally present in aliving system (e.g., a cell) in the vicinity of the [3+2] cycloaddition.Specific Cu(I) chelators are known in the art and include, but are notlimited to, neocuproine (Al-Sa'doni et al., Br. J. Pharmacol.121:1047-1050 (1997); De Man et al., Eur. J. Pharmacol. 381:151-159(1999); Gocmen et al., Eur. J. Pharmacol. 406: 293-300 (2000)) andbathocuproine disulphonate (Bagnati et al., Biochem. Biophys. Res.Commun. 253:235-240 (1998)). In other embodiments, an alternativecopper-free system that does not require a copper-chelating moiety isemployed. For example, [3+2] cycloadditions between azides andcyclooctynes and difluorocyclooctynes described by Bertozzi et al. (J.Am. Chem. Soc. 126:15046-15047 (2004)) may be employed.

Staudinger Ligation

In certain embodiments, methods provided herein generally include aStaudinger ligation. In certain embodiments, the Staudinger ligationoccurs between an azide group on a polypeptide-puromycin analogconjugate and a staining agent comprising an optionally substitutedtriarylphosphine attached to a label.

Optimization of reaction conditions for the Staudinger is within theskill in the art. In certain embodiments, the Staudinger ligation isperformed under aqueous conditions. Examples of reaction conditions havebeen described, for example, in: Saxon et al., Science, 2000, 287:2007-2010; Saxon et al., Org. Lett., 2000, 2: 2141-2143; Kiick et al.,Proc. Natl. Acad. Sci. USA, 2002, 99: 19-24; Lemieux et al., J. Am.Chem. Soc., 2003, 125: 4708-4709; Prescher et al., Nature, 2004, 430:873-877.

Other Bioorthogonal Reactions

Other bioorthogonal reactions may also be used in methods providedherein. In certain embodiments, methods provided herein generallyinclude a bioorthogonal reaction selected from the group consisting of[3+2] cycloaddition, Staudinger ligation, inverse electron demandDiels-Alder, oxime ligation, hydrazone ligation, and [2+2+2]cycloaddition. In certain embodiments, an inverse electron demandDiels-Alder reaction is used in a provided method. In certainembodiments, a tetrazine ligation is used in a provided method. Incertain embodiments, an oxime ligation is used in a method providedherein. In certain embodiments, a hydrazone ligation is used in a methodprovided herein. In certain embodiments, a [2+2+2] cycloaddition is usedin a method provided herein. In certain embodiments, a quadricyclaneligation is used in a method provided herein.

Labels and Detection of Labeled Polypeptides

In certain embodiments, methods of the present invention include abioorthogonal reaction between a first reactive group on a puromycinanalog conjugated to a polypeptide and a second reactive group attachedto a label. The bioorthogonal reaction results in labeling of thepolypeptide. In certain embodiments, methods of the present inventioninclude a [3+2] cycloaddition between a first reactive unsaturated groupon a puromycin analog conjugated to a polypeptide and a second reactiveunsaturated group attached to a label, resulting in labeling of thepolypeptide.

As described herein, the role of a label is to allow visualization,detection, and/or identification of a polypeptide, e.g., a nascentpolypeptide in a cell, following labeling. In certain embodiments, alabel (or detectable agent or moiety) is selected such that it generatesa signal which can be measured and whose intensity is related (e.g.,proportional) to the amount of labeled polypeptide, e.g., in a samplebeing analyzed. In certain embodiments, in array-based detection methodsdescribed herein, the detectable agent is also selected such that itgenerates a localized signal, thereby allowing spatial resolution of thesignal for each spot on the array.

In certain embodiments, the association between a label and a labelingreagent comprising the second reactive group (e.g., a reactiveunsaturated group) is covalent. A label can be directly attached to areactive group on the labeling reagent or indirectly through a linker.

Methods for attaching detectable moieties to chemical molecules arewell-known in the art. In certain embodiments, the label and unsaturatedgroup are directly, covalently linked to each other. In certainembodiments, the direct covalent binding is through an amide, ester,carbon-carbon, disulfide, carbamate, ether, thioether, urea, amine, orcarbonate linkage. In certain embodiments, covalent binding is achievedby taking advantage of functional groups present on the reactive groupand detectable moiety. Suitable functional groups that can be used toattach the two chemical entities together include, but are not limitedto, amines, anhydrides, hydroxy groups, carboxy groups, and thiols. Adirect linkage may also be formed using an activating agent, such as acarbodiimide. A wide range of activating agents are known in the art andare suitable for linking a label and an reactive group (e.g., a reactiveunsaturated group).

In other embodiments, a reactive group (e.g., a reactive unsaturatedgroup) of a labeling reagent and a label are indirectly covalentlylinked to each other via a linker group. Such indirect attachment can beaccomplished by using any number of stable bifunctional agents wellknown in the art, including homofunctional and heterofunctional linkers(see, for example, Pierce Catalog and Handbook). Use of a bifunctionallinker differs from the use of an activating agent in that the formerresults in a linking moiety being present in the reaction product,whereas the latter results in a direct coupling between the two moietiesinvolved in the reaction. The role of the bifunctional linker may be toallow the reaction between two otherwise inert moieties. Alternativelyor additionally, the bifunctional linker, which becomes part of thereaction product, may be selected such that it confers some degree ofconformational flexibility to the reaction product, or other useful ordesired properties. In certain embodiments, the linker is cleavable(e.g., chemically cleavable or photochemically cleavable). The presenceof a cleavable linker between the label and the puromycin analog allowsfor temporary labeling of the polypeptide-puromycin analog conjugate.With such a system, whenever desired (e.g., following detection of thepolypeptide), the label can be cleaved off the puromycin analog to whichit is attached. Cleavable linkers are known in the art. For example, thelinker may be a cystamine linker, the disulfide bond of which can bereduced using dithiothreitol (DTT).

Any of a wide variety of labeling/detectable agents can be used in thepractice of the present invention. Suitable detectable agents include,but are not limited to, various ligands, radionuclides (such as, forexample, ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵I, ¹³¹I, and the like); fluorescent dyes(for specific exemplary fluorescent dyes, see below); chemiluminescentagents (such as, for example, acridinium esters, stabilized dioxetanes,and the like); spectrally resolvable inorganic fluorescent semiconductornanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold,silver, copper and platinum) or nanoclusters; enzymes (such as, forexample, those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase); colorimetriclabels (such as, for example, dyes, colloidal gold, and the like);magnetic labels (such as, for example, Dynabeads™); and biotin,dioxigenin, haptens, and proteins for which antisera or monoclonalantibodies are available.

In certain embodiments, the label comprises a fluorescent moiety.Numerous known fluorescent labeling moieties of a wide variety ofchemical structures and physical characteristics are suitable for use inthe practice of the present invention. Suitable fluorescent dyesinclude, but are not limited to, fluorescein and fluorescein dyes (e.g.,fluorescein isothiocyanine or FITC, naphthofluorescein,4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6-carboxyfluorescein orFAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes,phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g.,carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G,rhodamine Green, rhodamine Red, tetramethylrhodamine or TMR), coumarinand coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes(e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red,Texas Red-X, Spectrum Red™, Spectrum Green™ cyanine dyes (e.g. Cy-3™,Cy-5™, Cy-3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, AlexaFluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, AlexaFluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPYdyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591,BODIPY 630/650, BODIPY 650/665), IRDyes (e.g., IRD40, IRD 700, IRD 800),and the like. For more examples of suitable fluorescent dyes and methodsfor coupling fluorescent dyes to other chemical entities see, forexample, “The Handbook of Fluorescent Probes and Research Products”, 9thEd., Molecular Probes, Inc., Eugene, Oreg.

Favorable properties of fluorescent labeling agents to be used in thepractice of the invention include high molar absorption coefficient,high fluorescence quantum yield, and photostability. In certainembodiments, labeling fluorophores desirably exhibit absorption andemission wavelengths in the visible (i.e., between 400 and 750 nm)rather than in the ultraviolet range of the spectrum (i.e., lower than400 nm). Other desirable properties of the fluorescent moiety mayinclude cell permeability and low toxicity, for example if labeling ofthe polypeptide is to be performed in a cell or an organism (e.g., aliving animal).

As reported in the Examples, various fluorescent labelling reagents(e.g., staining reagents) have been used by the present Applicants,including Alexa568-azide, which is non-cell permeable, andtetramethylrhodamine (TMR)-azide, which is cell permeable.

Selection of a particular label will depend on the purpose of thelabeling to be performed and will be governed by several factors, suchas the ease and cost of the labeling method, the quality of samplelabeling desired, the effects of the detectable moiety on the cell ororganism, the nature of the detection system, the nature and intensityof the signal generated by the detectable moiety, and the like.

As will be recognized by one of ordinary skill in the art, detection ofpolypeptides labeled according to methods disclosed herein may beperformed by any of a wide variety of methods, and using any of a widevariety of techniques. Selection of a suitable detection method and/ordetection technique based on the nature of the label (e.g.,radionuclide, fluorophore, chemiluminescent agent, quantum dot, enzyme,magnetic label, hapten, etc.) is within the skill in the art.

For example, fluorescently labeled polypeptides may be detected usingfluorescence detection techniques, including, but not limited to, flowcytometry and fluorescence microscopy. Selection of a specificfluorescence detection technique will be governed by many factorsincluding the purpose of the labeling experiment (e.g., study ofchromosomes ultrastructure, cell proliferation determination, ortoxicity assay) as well as the location of the labeled polypeptide to bedetected (i.e., such as inside a living cell or inside a tissue).

Flow cytometry is a sensitive and quantitative technique that analyzesparticles (such as cells) in a fluid medium based on the particles'optical characteristic (H. M. Shapiro, “Practical Flow Cytometry”, 3rdEd., 1995, Alan R. Liss, Inc.; and “Flow Cytometry and Sorting, SecondEdition”, Melamed et al. (Eds), 1990, Wiley-Liss: New York). A flowcytometer hydrodynamically focuses a fluid suspension of particlescontaining one or more fluorophores, into a thin stream so that theparticles flow down the stream in a substantially single file and passthrough an examination or analysis zone. A focused light beam, such as alaser beam, illuminates the particles as they flow through theexamination zone, and optical detectors measure certain characteristicsof the light as it interacts with the particles (e.g., light scatter andparticle fluorescence at one or more wavelengths).

Alternatively or additionally, fluorescently labeled polypeptides incells, tissues or organisms may be visualized and detected byfluorescence microscopy using various imaging techniques. In addition toconventional fluorescence microscopy, fluorescently labeled polypeptidescan be analyzed by, for example, time-lapse fluorescence microscopy,confocal fluorescence microscopy, or two-photon fluorescence microscopy.Time-lapse microscopy techniques (D. J. Stephens and V. J. Allan,Science, 2003, 300:82-86) can provide a complete picture of complexcellular processes that occur in three dimensions over time. Informationacquired by these methods allow dynamic phenomena such as cell growth,cell motion and cell nuclei division to be monitored and analyzedquantitatively. Confocal microscopy (L. Harvath, Methods Mol. Biol.,1999, 115:149-158; Z. Foldes-Papp et al., Int. Immunopharmacol., 2003,3:1715-1729) offers several advantages over conventional opticalmicroscopy, including controllable depth of field, the elimination ofimage degrading out-of-focus information, and the ability to collectserial optical sections from thick specimens (e.g., tissues or animals).Two-photon fluorescence microscopy (P. T. So et al., Annu. Rev. Biomed.Eng., 2000, 2:399-429), which involves simultaneous absorption of twophotons by the fluorophore at the focal point of the microscope, allowsthree-dimensional imaging in highly localized volumes (e.g., in thenucleus of cells) with minimal photobleaching and photodamage.

Signals from fluorescently labeled polypeptides attached to microarraysor located inside cells in multi-well plates can be detected andquantified by any of a variety of automated and/or high-throughputinstrumentation systems including fluorescence multi-well plate readers,fluorescence activated cell sorters (FACS) and automated cell-basedimaging systems that provide spatial resolution of the signal. Methodsfor the simultaneous detection of multiple fluorescent labels and thecreation of composite fluorescence images are well-known in the art andinclude the use of “array reading” or “scanning” systems, such ascharge-coupled devices (i.e., CCDs) (see, for example, Hiraoka et al.,Science, 1987, 238: 36-41; Aikens et al., Meth. Cell Biol. 1989,29:291-313; Divane et al., Prenat. Diagn. 1994, 14: 1061-1069; Jalal etal., Mayo Clin. Proc. 1998, 73: 132-137; Cheung et al., Nature Genet.1999, 21: 15-19; see also, for example, U.S. Pat. Nos. 5,539,517;5,790,727; 5,846,708; 5,880,473; 5,922,617; 5,943,129; 6,049,380;6,054,279; 6,055,325; 6,066,459; 6,140,044; 6,143,495; 6,191,425;6,252,664; 6,261,776; and 6,294,331). A variety of instrumentationsystems have been developed to automate such analyses including theautomated fluorescence imaging and automated microscopy systemsdeveloped by Cellomics, Inc. (Pittsburgh, Pa.), Amersham Biosciences(Piscataway, N.J.), TTP LabTech Ltd (Royston, UK), Quantitative 3Dimensional Microscopy (Q3DM) (San Diego, Calif.), Evotec AG (Hamburg,Germany), Molecular Devices Corp. (Sunnyvale, Calif.), and Carl Zeiss AG(Oberkochen, Germany).

Signal-to-Noise Ratio Improvements

In another aspect, the present invention provides a system for improvingthe signal-to-noise ratio in the detection of a polypeptide labeled witha fluorescent moiety using a labeling process disclosed herein.

Any molecule of labeling reagent that has not been consumed by abioorthogonal labeling reaction may contribute to the background (i.e.,non-specific) signal. The present invention provides a strategy forreducing or eliminating this background signal which comprises quenchingthe fluorescent signal of the label on the unreacted labeling reagent byreaction with a molecule comprising a quencher moiety. For example, insome embodiments, the reaction between the reagent and the moleculecomprising the quencher moiety is a [3+2] cycloaddition.

Thus, certain inventive methods for improving the signal-to-noise ratioin detection of a fluorescently labeled polypeptide prepared asdescribed herein, comprise contacting unreacted reagent comprising asecond reactive unsaturated group and a fluorescent label with aquenching molecule comprising a reactive unsaturated group attached to aquenching moiety such that a [3+2] cycloaddition takes place between thereactive unsaturated groups of the labelling reagent and quenchingmolecule. After reaction, the physical proximity between the fluorescentlabel and the quenching moiety prevents detection of a fluorescentsignal from the fluorescent label.

Examples of quenching moieties include, but are not limited to, DABCYL(i.e., 4-(4′-dimethylaminophenylazo)-benzoic acid) succinimidyl ester,diarylrhodamine carboxylic acid, succinimidyl ester (or QSY-7), and4′,5′-dinitrofluorescein carboxylic acid, succinimidyl ester (or QSY-33)(all available, for example, from Molecular Probes), quencher1 (Q1;available from Epoch Biosciences, Bothell, Wash.), or “Black holequenchers” BHQ-1, BHQ-2, and BHQ-3 (available from BioSearchTechnologies, Inc., Novato, Calif.).

Labeling of Polypeptides in Cells

The present invention also provides methods for labeling polypeptides incells. In some embodiments, such methods comprise: contacting a cellwith an effective amount of a puromycin analog comprising a firstreactive group, such that the puromycin analog is covalently bound tothe C-terminus of one or more nascent polypeptides in the cell to formone or more polypeptide-puromycin analog conjugates; and contacting thecell with a compound comprising a second reactive group and a label,such that a bioorthogonal reaction occurs between the first and secondreactive groups. In certain embodiments, such methods comprise:contacting a cell with an effective amount of a puromycin analogcomprising a first reactive unsaturated group, such that the puromycinanalog is covalently bound to the C-terminus of one or more nascentpolypeptides in the cell to form one or more polypeptide-puromycinanalog conjugates; and contacting the cell with a compound comprising asecond reactive unsaturated group and a label, such that a [3+2]cycloaddition occurs between the first and second reactive unsaturatedgroups.

Unless otherwise stated, the labeling reagent and reaction conditionsused in these methods are analogous to those described above for themethods of labeling polypeptides. As discussed herein, the labelingmethods of the present invention exhibit several advantages overcurrently available labeling protocols including the possibility ofstaining polypeptides in living cells. The terms “living cell” and “livecell” are used herein interchangeably and refer to a cell which isconsidered living according to standard criteria for that particulartype of cell, such as maintenance of normal membrane potential, energymetabolism, or proliferative capability. In particular, the methods ofthe present invention do not require fixation and/or denaturation of thecells.

In some embodiments, the invention relates to incorporation of labelsinto polypeptides in cells in culture. In certain embodiments, the cellsare grown in standard tissue culture plastic ware. Such cells includenormal and transformed cells derived. In certain embodiments, cells areof mammalian (human or animal, such as rodent or simian) origin.Mammalian cells may be of any fluid, organ or tissue origin (e.g.,blood, brain, liver, lung, heart, bone, and the like) and of any celltypes (e.g., basal cells, epithelial cells, platelets, lymphocytes,T-cells, B-cells, natural killer cells, macrophages, tumor cells, andthe like).

Cells suitable for use in the methods of the present invention may beprimary cells, secondary cells or immortalized cells (i.e., establishedcell lines). They may have been prepared by techniques well-known in theart (for example, cells may be obtained by drawing blood from a patientor a healthy donor) or purchased from immunological and microbiologicalcommercial resources (for example, from the American Type CultureCollection, Manassas, Va.). Alternatively or additionally, cells may begenetically engineered to contain, for example, a gene of interest suchas a gene expressing a growth factor or a receptor.

Cells to be used in the methods of the present invention may be culturedaccording to standard culture techniques. For example, cells are oftengrown in a suitable vessel in a sterile environment at 37° C. in anincubator containing a humidified 95% air-5% CO₂ atmosphere. Vessels maycontain stirred or stationary cultures. Various cell culture media maybe used including media containing undefined biological fluids such asfetal calf serum. Cell culture techniques are well known in the art, andestablished protocols are available for the culture of diverse celltypes (see, for example, R. I. Freshney, Culture of Animal Cells: AManual of Basic Technique, 2nd Edition, 1987, Alan R. Liss, Inc.).

Incorporation of Puromycin Analog into Nascent Polypeptides to FormConjugates

Puromycin-polypeptide conjugates are well known in the art. Puromycin isan antibiotic that competes with aminoacyl-tRNAs for the A-site ofribosomes. After binding to the ribosome, puromycin then becomescovalently attached to the C-terminus of the nascent polypeptide chain,resulting in termination. The puromycin mechanism of action is used toprovide a snapshot of nascent polypeptides synthesized in vivo.Traditional methods include using a polyclonal antibody to puromycin toisolate the conjugates (Hansen et al., J. Biol. Chem. 269:26610-26613(1994)) or employing bioorthogonal methionine (Met) analogs such as anazido analog azidohomoalanine (Aha) and an alkyne analoghomopropargylglycine (Hpg) (Dietrich et al., Proc. Natl. Acad. Sci. USA103:9482-9487 (2006); Beatty et al., Angew. Chem. Intl. Ed. Engl.45:7364-7367 (2006)). However, there are disadvantages to thetraditional approaches. Antibod-based technologies can exhibit a lot ofvariability and non-specific background noise. Antibodies are lessuniform and more difficult to standardize than a chemical compound.Antibodies have high molecular weights and do not penetrate effectivelyand efficiently through a thick tissue or organ sample. In contrast,fluorescent molecules such as those employed in the present inventionare much smaller than antibodies, in some embodiments about 300-500times smaller, and in some embodiments they can diffuse into and stainthick specimens. In some embodiments, the methods described herein allowwhole-mount fluorescent imaging of large fragments of tissue and organs,which would have to be physically sectioned to be imaged by traditionalimmunofluorescence. Metabolic labeling with Met analogs requiresMet-free media, which prevents the use of this method in animals, unlikethe methods of the present invention which can be used in vivo. Metanalog incorporation is proportional to the number of Met residues in aprotein, while the puromycin analogs of the present inventionincorporate at exactly one molecule per nascent polypeptide chain. Metanalogs will not label proteins that do not start with or contain a Metresidue, while the methods of the present invention do not depend onamino acid content. Met analogs need to be first activated and convertedto amino acyl-tRNAs before incorporation into proteins; by contrast,puromycin analogs of the present invention generate covalent conjugateswith nascent polypeptide chains directly, without any priormodification.

Contacting cells in vitro with an effective amount of a puromycin analogsuch that the puromycin analog is conjugated to a nascent polypeptide inthe cell may be carried out using any suitable protocol. A step ofcontacting a cell with an effective amount of a puromycin analog may beperformed, for example, by incubating the cell with the puromycin analogunder suitable incubation conditions (e.g., in culture medium at 37°C.). In certain embodiments, it may be desirable to avoid disturbing thecells in any way (e.g., by centrifugation steps or temperature changes)that may perturb their protein synthesis patterns. The incubation timewill be dependent on the cell population's rate of cell cycling entryand progression. Optimization of incubation time and conditions iswithin the skill in the art.

Following conjugation of a puromycin analog to a nascent polypeptides ofin vitro cells, the step of contacting the cells with a labeling reagentcomprising the second reactive group and a label may be performed by anysuitable method. In some embodiments, cells are incubated in thepresence of a labeling reagent in a suitable incubation medium (e.g.,culture medium) at 37° C. and for a time sufficient for the reagent topenetrate into the cell and react with any polypeptide-puromycin analogconjugate. Optimization of the concentration of labeling reagent,reaction time and conditions is within the skill in the art.

As described herein, in embodiments where the presence of exogenousCu(I) is not desirable, a [3+2] cycloaddition may be carried out usingvarious copper-free reaction conditions.

In embodiments where the labelling reagent does not exhibit high cellpermeability, permeabilization may be performed to facilitate access ofthe labelling reagent to cellular cytoplasm, or intracellular componentsor structures of the cells. In certain embodiments, permeabilization mayallow a reagent to enter into a cell and reach a concentration withinthe cell that is greater than that which would normally penetrate intothe cell in the absence of such permeabilization treatment.

Permeabilization of the cells may be performed by any suitable method(see, for example, C. A. Goncalves et al., Neurochem. Res. 2000, 25:885-894). Such methods include, but are not limited to, exposure to adetergent (such as CHAPS, cholic acid, deoxycholic acid, digitonin,n-dodecyl-β-D-maltoside, lauryl sulfate, glycodeoxycholic acid,n-lauroylsarcosine, saponin, and triton X-100) or to an organic alcohol(such as methanol and ethanol). Other permeabilization methods comprisethe use of certain peptides or toxins that render membranes permeable(see, for example, O. Aguilera et al., FEBS Lett. 1999, 462: 273-277; A.Bussing et al., Cytometry, 1999, 37: 133-139). Selection of anappropriate permeabilizing agent and optimization of the incubationconditions and time can easily be performed by one of ordinary skill inthe art.

Labeling of Polypeptides in Tissues or Organisms

The present invention also provides methods for labeling polypeptides inorganisms (i.e., living biological systems). In certain embodiments,such methods comprise the steps of administering to an organism aneffective amount of a puromycin analog comprising a first reactivegroup, such that the puromycin analog is covalently bound to theC-terminus of one or more nascent polypeptides in the organism to formone or more polypeptide-puromycin analog conjugates; contacting at leastone cell of the organism with a compound comprising a second reactivegroup and a label, such that a bioorthogonal reaction occurs between thefirst and second reactive groups. In certain embodiments, a providedmethod comprises the steps of administering to an organism an effectiveamount of a puromycin analog comprising a first reactive unsaturatedgroup, such that the puromycin analog is covalently bound to theC-terminus of one or more nascent polypeptides in the organism to formone or more polypeptide-puromycin analog conjugates; contacting at leastone cell of the organism with a compound comprising a second reactiveunsaturated group and a label, such that a [3+2] cycloaddition occursbetween the first and second reactive unsaturated groups.

Unless otherwise stated, labelling reagents and reaction conditions usedin these methods are analogous to those described herein for the methodsof labeling polypeptides in cells and can be determined/optimized by oneskilled in the art.

Methods of labeling of the present invention may be performed using anyliving system that has or can develop the ability to act or functionindependently. Thus, labeling methods of the present invention may beperformed in unicellular or multicellular systems, including, humans,animals, plants, bacteria, protozoa, and fungi. In certain embodiments,labeling methods of the present invention are performed in a human oranother mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). In certain embodiments, labeling methods ofthe present invention are performed in a non-human whole animal.

Administration of a puromycin analog to an organism may be performedusing any suitable method that results in conjugation of the puromycinanalog to nascent polypeptides of the organism.

For example, a puromycin analog may be formulated in accordance withconventional methods in the art. Proper formulation is dependent uponthe route of administration chosen. Suitable routes of administrationcan, for example, include oral, rectal, transmucosal, transcutaneous, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Alternatively, a puromycin analog preparationcan be administered in a local rather than systemic manner, for example,via injection directly into a specific tissue, often in a depot orsustained release formulation.

Following conjugation of a puromycin analog to nascent polypeptides incells of the organism, a step of contacting at least one cell of theorganism with a reagent comprising the second reactive group attached toa label may be performed by any suitable method that allows for abioorthogonal reaction to take place.

In certain embodiments, cells are collected (e.g., by drawing blood fromthe organism), isolated from a tissue obtained by biopsy (e.g., needlebiopsy, laser capture micro dissection or incisional biopsy) or isolatedfrom an organ or part of an organ (e.g., harvested at autopsy). Thecells can then be submitted to the bioorthogonal labeling as describedabove.

In other embodiments, a tissue obtained by biopsy or an organ or part ofan organ harvested at autopsy may be prepared for labelling as known inthe art (e.g., fixed, embedded in paraffin and sectioned) and incubatedin the presence of the bioorthogonal reagent (e.g., after de-waxing).

Example 4 describes an experiment where a mouse was intraperitoneallyinjected with OP-puro and its organs harvested 1 hour after injection,prepared for staining and stained with tetramethylrhodamine (TMR)-azideand with Hoechst. Fluorescence images of the small intestine, spleen,kidney, and liver are presented in FIGS. 3 and 4.

Isolated Labeled Polypeptides

In another aspect, the present invention provides isolated detectablepolypeptides, for example, prepared by one of the methods describedherein. More specifically, in certain embodiments, the present inventionprovides polypeptides that are detectable following a bioorthogonalreaction as well as isolated polypeptides that contain at least onedetectable moiety that has been incorporated via a bioorthogonalreaction. In certain embodiments, the present invention providespolypeptides that are detectable following a [3+2] cycloadditionreaction as well as isolated polypeptides that contain at least onedetectable moiety that has been incorporated via a [3+2] cycloaddition.

In certain embodiments, an inventive polypeptide contains at least onepuromycin analog comprising a reactive group. In certain embodiments,the reactive group undergoes a bioorthogonal reaction in the presence ofa reagent comprising a different reactive group attached to a label. Incertain embodiments, an inventive polypeptide contains at least onepuromycin analog comprising a reactive unsaturated group. In certainembodiments, the reactive unsaturated group undergoes a [3+2]cycloaddition in the presence of a reagent comprising a differentreactive unsaturated group attached to a label.

In other embodiments, an inventive polypeptide contains at least onepuromycin analog attached to a label. For example, the puromycin analogmay comprise a cycloadduct resulting from a [3+2] cycloaddition.

Detectable polypeptides of the present invention may be prepared by anysuitable method, as described herein, including synthetic methods,enzymatic methods, and by using ribosomal machinery.

As can be appreciated by one of ordinary skill in the art, isolateddetectable polypeptides of the present invention may be used in a widevariety of applications. For example, isolated detectable polypeptidesmay be used in assays. In certain embodiments, detectable polypeptidesmay be provided with appropriate labelling reagents. In otherembodiments, detectable polypeptides are provided attached to an arrayor micro-array.

Arrays according to the present invention comprise a plurality ofdetectable polypeptides immobilized to discrete spots on a substratesurface. Substrate surfaces can be made of any of rigid, semi-rigid orflexible materials that allow for direct or indirect attachment (i.e.,immobilization) of detectable polypeptides to the substrate surface.Suitable materials include, but are not limited to, cellulose, celluloseacetate, nitrocellulose, glass, quartz other crystalline substrates suchas silicones, and various plastics and plastic copolymers. Whenfluorescence is to be detected, arrays comprising cyclo-olefin polymersmay be used in some embodiments.

The presence of reactive functional chemical groups on the materials canbe exploited to directly or indirectly attach the detectablepolypeptides to the substrate surface. Methods for immobilizingpolypeptides to substrate surfaces to form an array are well-known inthe art.

Cells Comprising Labeled Polypeptides

In another aspect, the present invention provides cells comprisingdetectable polypeptides, for example prepared by one or more of themethods described herein. More specifically, in certain embodiments, thepresent invention provides cells comprising polypeptides that aredetectable following a bioorthogonal reaction as well as cellscomprising polypeptides that contain at least one detectable moiety thathas been incorporated via a bioorthogonal reaction. In certainembodiments, the present invention provides cells comprisingpolypeptides that are detectable following a [3+2] cycloadditionreaction as well as cells comprising polypeptides that contain at leastone detectable moiety that has been incorporated via a [3+2]cycloaddition.

As will be recognized by one of ordinary skill in the art, a cell of thepresent invention may comprise any of the detectable polypeptidesdescribed herein.

Determination of Protein Synthesis

In another aspect, the present invention provides methods for measuringprotein synthesis and/or protein synthesis rates in a cell or anorganism. Such methods may comprise steps of: contacting a cell with aneffective amount of a puromycin analog comprising a first reactivegroup, such that the puromycin analog is covalently bound to theC-terminus of one or more nascent polypeptides in the cell to form oneor more polypeptide-puromycin analog conjugates; contacting the cellwith a compound comprising a second reactive group and a label, suchthat a bioorthogonal reaction occurs between the first and secondreactive groups; and determining the amount of labeled protein in thecell to measure protein synthesis. In certain embodiments, a providedmethod comprises steps of: contacting a cell with an effective amount ofa puromycin analog comprising a first reactive unsaturated group, suchthat the puromycin analog is covalently bound to the C-terminus of oneor more nascent polypeptides in the cell to form one or morepolypeptide-puromycin analog conjugates; contacting the cell with acompound comprising a second reactive unsaturated group and a label,such that a [3+2] cycloaddition occurs between the first and secondreactive unsaturated groups; and determining the amount of labeledprotein in the cell to measure protein synthesis. In certainembodiments, the amount of label gives information about the extent ofprotein synthesis. In other embodiments, the amount of label givesinformation about the rate of protein synthesis.

In other embodiments, such methods comprise steps of: administering toan organism an effective amount of a puromycin analog comprising a firstreactive group, such that the puromycin analog is covalently bound tothe C-terminus of one or more nascent polypeptides in the organism toform one or more polypeptide-puromycin analog conjugates; contacting atleast one cell of the organism with a compound comprising a secondreactive group and a label, such that a bioorthogonal reaction occursbetween the first and second reactive groups; and determining the amountof label in the at least one cell in order to measure protein synthesisin the organism. In certain embodiments, a provided method comprisessteps of: administering to an organism an effective amount of apuromycin analog comprising a first reactive unsaturated group, suchthat the puromycin analog is covalently bound to the C-terminus of oneor more nascent polypeptides in the organism to form one or morepolypeptide-puromycin analog conjugates; contacting at least one cell ofthe organism with a compound comprising a second reactive unsaturatedgroup and a label, such that a [3+2] cycloaddition occurs between thefirst and second reactive unsaturated groups; and determining the amountof label in the at least one cell in order to measure protein synthesisin the organism.

In certain embodiments, the amount of label gives information about theextent of protein synthesis in the organism. In other embodiments, theamount of label gives information about the rate of protein synthesis inthe organism.

These methods may be performed using techniques and procedures asdescribed herein for methods of labeling polypeptides in cells andorganisms. With such methods, the manner of performing the contactingand/or administering steps, type of labelling reagent, type of label,and techniques for the detection of such labels are analogous to thosedescribed for other methods of the present invention relating tolabeling polypeptides in cells or in organisms.

Methods for measuring protein synthesis or protein synthesis ratesaccording to the present invention may be used in a wide variety ofapplications, including, but not limited to characterization of celllines, optimization of cell culture conditions, characterization ofprotein synthesis in normal, diseased and injured tissues, and diagnosisof a variety of diseases and disorders in which protein synthesis isinvolved. In certain embodiments, methods of the present invention allowfor the identification of proteins regulated at the level oftranslation, such as targets of specific miRNAs, targets of theRNA-binding proteins that control translation of specific mRNAs, andtargets of signaling pathways that regulate translation, such as TORsignaling.

Diseases and disorders characterized by altered rates of proteinsynthesis can be monitored by methods of the present invention.

Screening Assays

In another aspect, the present invention provides methods for theidentification of agents that perturb protein synthesis. These methodsmay be used for screening agents for their ability to induce (i.e.,increase, enhance or otherwise exacerbate) or inhibit (i.e., decrease,slow down or otherwise suppress) protein synthesis.

For example, such methods may comprise steps of: contacting a cell witha test agent; contacting the cell with an effective amount of apuromycin analog comprising a first reactive group, such that thepuromycin analog is covalently bound to the C-terminus of nascentpolypeptides in the cell; contacting the cell with a compound comprisinga second reactive group and a label, such that a bioorthogonal reactionoccurs between the first and second reactive groups; determining theamount of label incorporated into the nascent polypeptides, wherein theamount of label indicates the extent of protein synthesis; andidentifying the test agent as an agent that perturbs cellularproliferation if the amount of label incorporated into nascentpolypeptides is less than or greater than the amount of label measuredin a control in which a cell is not contacted with the test agent. Incertain embodiments, a provided method comprises steps of: contacting acell with a test agent; contacting the cell with an effective amount ofa puromycin analog comprising a first reactive unsaturated group, suchthat the puromycin analog is covalently bound to the C-terminus ofnascent polypeptides in the cell; contacting the cell with a compoundcomprising a second reactive unsaturated group and a label, such that a[3+2] cycloaddition occurs between the first and second reactiveunsaturated groups; determining the amount of label incorporated intothe nascent polypeptides, wherein the amount of label indicates theextent of protein synthesis; and identifying the test agent as an agentthat perturbs cellular proliferation if the amount of label incorporatedinto nascent polypeptides is less than or greater than the amount oflabel measured in a control in which a cell is not contacted with thetest agent.

In certain embodiments, the determining step is limited to the portionof the proteome being synthesized upon contacting with a test agent.

The manner of performing the steps of contacting the cell; the labellingreagent; the label type; and methods of detecting the labeledpolypeptides are analogous to those described for other methods of thepresent invention relating to measuring protein synthesis and proteinsynthesis rates in cells in vitro.

As will be appreciated by one of ordinary skill in the art, screeningmethods of the present invention may also be used to identify compoundsor agents that regulate protein synthesis.

In certain embodiments, screening assays of the present invention may beperformed using any normal or transformed cells that can be grown instandard tissue culture plastic ware. Cells may be primary cells,secondary cells, or immortalized cells. In certain embodiments, cells tobe used in inventive screening methods are of mammalian (human oranimal) origin. Cells may be from any organ or tissue origin and of anycell types, as described above.

Selection of a particular cell type and/or cell line to perform ascreening assay according to the present invention will be governed byseveral factors such as the nature of the agent to be tested and theintended purpose of the assay. For example, a toxicity assay developedfor primary drug screening may be performed using established celllines, which are commercially available and usually relatively easy togrow, while a toxicity assay to be used later in the drug developmentprocess may preferably be performed using primary or secondary cells,which are often more difficult to obtain, maintain, and/or grow thanimmortalized cells but which represent better experimental models for invivo situations.

In certain embodiments, screening methods are performed using cellscontained in a plurality of wells of a multi-well assay plate. Suchassay plates are commercially available, for example, from StrategeneCorp. (La Jolla, Calif.) and Corning Inc. (Acton, Mass.), and include,for example, 48-well, 96-well, 384-well and 1536-well plates.

As will be appreciated by those of ordinary skill in the art, any kindof compounds or agents can be tested using inventive methods. A testcompound may be a synthetic or natural compound; it may be a singlemolecule, a mixture of different molecules or a complex of differentmolecules. In certain embodiments, inventive methods are used fortesting one or more compounds. In other embodiments, inventive methodsare used for screening collections or libraries of compounds.

Compounds that can be tested for their capacity or ability to perturb(i.e., induce or inhibit) or regulate protein synthesis may belong toany of a variety of classes of molecules including, but not limited to,small molecules, peptides, saccharides, steroids, antibodies (includingfragments or variants thereof), fusion proteins, antisensepolynucleotides, ribozymes, small interfering RNAs, peptidomimetics, andthe like.

Compounds or agents to be tested according to methods of the presentinvention may be known or suspected to perturb or regulate proteinsynthesis. Alternatively, assays may be performed using compounds oragents whose effects on protein synthesis are unknown.

Examples of compounds that may affect protein synthesis and that can betested by the methods of the present invention include, but are notlimited to, carcinogens; toxic agents; chemical compounds such assolvents; mutagenic agents; pharmaceuticals; particulates, gases andnoxious compounds in smoke (including smoke from cigarette, cigar andindustrial processes); food additives; biochemical materials; hormones;pesticides; ground-water toxins; and environmental pollutants. Examplesof agents that may affect protein synthesis and that can be tested bythe methods of the present invention include, but are not limited to,microwave radiation, electromagnetic radiation, radioactive radiation,ionizing radiation, heat, and other hazardous conditions produced by orpresent in industrial or occupational environments.

According to screening methods of the present invention, determinationof the ability of a test agent to perturb or regulate protein synthesisincludes comparison of the amount of label incorporated intopolypeptides of a cell that has been contacted with the test agent withthe amount of label incorporated into polypeptides of a cell that hasnot been contacted with the test agent.

A test agent is identified as an agent that perturbs protein synthesisif the amount of label incorporated into polypeptides of the cell thathas been contacted with the test agent is less than or greater than theamount of label measured in the control cell. More specifically, if theamount of label incorporated into polypeptides of the cell that has beencontacted with the test agent is less than the amount of label measuredin the control cell, the test agent is identified as an agent thatinhibits protein synthesis. If the amount of label incorporated intopolypeptides of the cell that has been contacted with the test agent isgreater than the amount of label measured in the control cell, the testagent is identified as an agent that induces protein synthesis.

Reproducibility of the results may be tested by performing the analysismore than once with the same concentration of the test agent (forexample, by incubating cells in more than one well of an assay plate).Additionally, since a test agent may be effective at varyingconcentrations depending on the nature of the agent and the nature of itmechanism(s) of action, varying concentrations of the test agent may betested (for example, added to different wells containing cells).Generally, test agent concentrations from 1 fM to about 10 mM are usedfor screening. In certain embodiments, screening concentrations arebetween about 10 pM and about 100 μM.

In certain embodiments, the methods described herein further involve theuse of one or more negative or positive control compounds. A positivecontrol compound may be any molecule or agent that is known to perturb(i.e., induce or inhibit) or regulate protein synthesis. A negativecontrol compound may be any molecule or agent that is known to have nodetectable effects on protein synthesis. In certain embodiments,inventive methods further comprise comparing the effects of the testagent to the effects (or absence thereof) of the positive or negativecontrol compound.

As will be appreciated by those skilled in the art, it is generallydesirable to further characterize an agent identified by the inventivescreening methods as an agent that perturbs or an agent that regulatesprotein synthesis. For example, if a test compound has been identifiedas an agent that perturbs (or regulates) protein synthesis using a givencell culture system (e.g., an established cell line), it may bedesirable to test this ability in a different cell culture system (e.g.,primary or secondary cells).

Test agents identified by screening methods of the present invention mayalso be further tested in assays that allow for the determination of theagents' properties in vivo. Accordingly, the present invention providesmethods for identifying an agent that perturbs protein synthesis orprotein synthesis rate in vivo. Such methods comprise steps of: exposingan organism to a test agent; administering to the organism an effectiveamount of a puromycin analog comprising a first reactive group, suchthat the puromycin analog is covalently bound to the C-terminus ofnascent polypeptides in the organism; contacting at least one cell ofthe organism with a compound comprising a second reactive group and alabel, such that a bioorthogonal reaction occurs between the first andsecond reactive groups; determining the amount of label incorporatedinto the nascent polypeptides in the at least one cell, wherein theamount of label indicates the extent of protein synthesis; andidentifying the test agent as an agent that perturbs cellularproliferation if the amount of label incorporated into nascentpolypeptides is less than or greater than the amount of label measuredin a control in which an organism is not administered a test agent. Incertain embodiments, a provided method comprises steps of: exposing anorganism to a test agent; administering to the organism an effectiveamount of a puromycin analog comprising a first reactive unsaturatedgroup, such that the puromycin analog is covalently bound to theC-terminus of nascent polypeptides in the organism; contacting at leastone cell of the organism with a compound comprising a second reactiveunsaturated group and a label, such that a [3+2] cycloaddition occursbetween the first and second reactive unsaturated groups; determiningthe amount of label incorporated into the nascent polypeptides in the atleast one cell, wherein the amount of label indicates the extent ofprotein synthesis; and identifying the test agent as an agent thatperturbs cellular proliferation if the amount of label incorporated intonascent polypeptides is less than or greater than the amount of labelmeasured in a control in which an organism is not administered a testagent.

As will be appreciated by one of ordinary skill in the art, thesemethods can be used to identify agents that regulate protein synthesisin vivo.

The manner of administration, labelling reagent, type of label andmethod of detection of the labeled polypeptides are analogous to thosedescribed herein for other inventive methods relating to measuringprotein synthesis in living systems.

Identification of Nascent Polypeptides

In another aspect, the present invention provides methods of isolatingand/or identifying nascent polypeptides.

For example, in certain embodiments, the present invention provides amethod of isolating a nascent polypeptide comprising contacting a cellwith an effective amount of a puromycin analog comprising a firstreactive group, such that the puromycin analog is covalently bound tothe C-terminus of a nascent polypeptide in the cell to form apolypeptide-puromycin analog conjugate; contacting the cell with acompound comprising a second reactive group and an affinity label, suchthat a bioorthogonal reaction occurs between the first and secondreactive groups to form an affinity-labeled polypeptide; and affinitypurifying the affinity-labeled polypeptide. In certain embodiments, thepresent invention provides a method of isolating a nascent polypeptidecomprising contacting a cell with an effective amount of a puromycinanalog comprising a first reactive unsaturated group, such that thepuromycin analog is covalently bound to the C-terminus of a nascentpolypeptide in the cell to form a polypeptide-puromycin analogconjugate; contacting the cell with a compound comprising a secondreactive unsaturated group and an affinity label, such that a [3+2]cycloaddition occurs between the first and second reactive unsaturatedgroups to form an affinity-labeled polypeptide; and affinity purifyingthe affinity-labeled polypeptide.

In certain embodiments, an affinity label comprises a hapten. In certainembodiments, the hapten is biotin.

In certain embodiments, the labeled polypeptide is identified by methodsknown in the art (e.g., mass spectrometry). Such methods are useful inidentifying proteins that are synthesized under certain conditions inthe cell. In some embodiments, the cell is in a whole animal.

In other embodiments, the present invention provides a method ofidentifying a nascent polypeptide comprising contacting a cell with aneffective amount of a puromycin analog comprising a first reactivegroup, such that the puromycin analog is covalently bound to theC-terminus of one or more nascent polypeptides in the cell to form oneor more polypeptide-puromycin analog conjugates; contacting the cellwith a solid support comprising a second reactive group, such that abioorthogonal reaction occurs between the first and second reactivegroups; and identifying the nascent polypeptide. In certain embodiments,the present invention provides a method of identifying a nascentpolypeptide comprising contacting a cell with an effective amount of apuromycin analog comprising a first reactive unsaturated group, suchthat the puromycin analog is covalently bound to the C-terminus of oneor more nascent polypeptides in the cell to form one or morepolypeptide-puromycin analog conjugates; contacting the cell with asolid support comprising a second reactive unsaturated group, such thata [3+2] cycloaddition occurs between the first and second reactiveunsaturated groups; and identifying the nascent polypeptide. In certainembodiments, a method of identifying further comprises cleaving apolypeptide from a surface.

In certain embodiments, the step of identifying is performed usingmethods known in the art (e.g., mass spectrometry). In otherembodiments, the step of contacting a cell is performed by administeringa puromycin analog to a whole animal, and at least one cell is thenisolated from the animal in order to perform subsequent steps.

Kits

In another aspect, the present invention provides kits comprisingmaterials useful for carrying out one or more of the methods describedherein. The inventive kits may be used by diagnostic laboratories,clinical laboratories, experimental laboratories, or practitioners. Theinvention provides kits which can be used in these different settings.

Basic materials and reagents for labeling polypeptides according to thepresent invention may be assembled together in a kit. An inventive kitfor labeling a polypeptide may include a puromycin analog as describedherein and a label. In certain embodiments, an inventive kit forlabeling a polypeptide includes a puromycin analog comprising a firstreactive group and a compound comprising a second reactive group and alabel. In certain embodiments, an inventive kit for labeling apolypeptide may include a puromycin analog comprising a first reactiveunsaturated group; and a compound comprising a second reactiveunsaturated group and a label. In certain embodiments, a kit comprisesreagents which render the procedure specific. Thus, if the detectableagent is a hapten, in certain embodiments the kit comprises thecorresponding appropriate antibody. Similarly, in certain embodiments akit intended to be used for the labeling of polypeptides in livingorganisms will contain puromycin analog formulated such that it can beadministered to a living organism. In certain embodiments, a kitintended to be used for screening compounds for their ability to induceor inhibit protein synthesis may include cells comprising labeledpolypeptides of the present invention.

Certain inventive kits may further comprise buffers and/or reagentsuseful to perform a bioorthogonal reaction. In certain embodiments, akit further comprises buffers and/or reagents useful to perform a [3+2]cycloaddition reaction, such as aqueous medium and Cu(I).

An inventive kit may further comprise one or more of: wash buffersand/or reagents, cell fixation buffers and/or reagents,immunohistochemical buffers and/or reagents, DAB photoconversion buffersand/or reagents, and detection means. In certain embodiment, the buffersand/or reagents are optimized for the particular labeling/detectiontechnique for which the kit is intended. Protocols for using thesebuffers and reagents for performing different steps of the procedure mayalso be included in the kit.

In certain embodiments, a kit according to the present inventioncontains instruments (e.g., needle biopsy syringe) and/or reagents forthe isolation of cells from an organism.

Reagents may be supplied in a solid (e.g., lyophilized) or liquid form.In certain embodiments, kits of the present invention comprise differentcontainers (e.g., vial, ampoule, test tube, flask or bottle) for eachindividual buffer and/or reagent. Each component will generally besuitable as aliquoted in its respective container or provided in aconcentrated form. Other containers suitable for conducting certainsteps of the labeling/detection assay may also be provided. In certainembodiments, individual containers of a kit are maintained in closeconfinement for commercial use.

In certain embodiments, a kit according to the present invention furthercomprises instructions for use. Instructions for using a kit accordingto one or more inventive methods may comprise instructions for labelingpolypeptides, instructions for measuring protein synthesis, instructionsfor interpreting results obtained as well as a notice in the formprescribed by a governmental agency (e.g., FDA) regulating themanufacture, use or sale of pharmaceuticals or biological products.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES General Methods

All solvents and reagents were obtained from commercial suppliers andwere used without further purification. NMR spectra were recorded on aVarian Oxford AS600 600 MHz NMR instrument. NMR chemical shifts wereexpressed in ppm relative to internal solvent peaks, and couplingconstants were measured in Hz. (br=broad). Mass spectra were determinedon a Waters Micromass ZQ instrument, using an ESI source coupled to aWaters 2525 HPLC system operating in reverse mode, with an WatersSunfire™ C18 5 μM 4.6×50 mm column. Flash chromatography was performedusing a Biotage Isolera One flash purification system.

Example 1 Synthesis of O-propargyl-puromycin

O-Propargyl Boc-Tyr-OMe (1)

A solution of Boc-Tyr-OMe (2.01 g, 6.80 mmol), propargyl bromide (80% wtsolution in toluene, 910 μL, 8.16 mmol), K₂CO₃ (2.82 g, 20.4 mmol) indry DMF (19 mL) was stirred for 17 h at room temperature. After dilutionwith water (200 mL), the resulting mixture was extracted with EtOAc(2×100 mL). The combined organic layers were washed with saturatedNaHCO₃ and brine followed by drying over Na₂SO₄. After removal of thesolvent in vacuo, the crude product was obtained as a yellow liquid andwas used in the next step without further purification.

O-Propargyl Boc-Tyr-OH (2)

A solution of O-Propargyl Boc-Tyr-OMe 1 (2.27 g, 6.80 mmol) wasdissolved in 27 mL 1,2-dichloroethane and after addition of trimethyltinhydroxide (3.69 g, 20.4 mmol), the mixture was heated to 80° C. untilTLC analysis indicated a complete reaction. The mixture was thenconcentrated in vacuo, and the residue was dissolved in EtOAc (100 mL).The organic layer was washed with aqueous HCl (5%) (3×60 mL), thenwashed with brine and dried over Na₂SO₄. After removal of the solvent invacuo, the residue was purified by flash column chromatography (SiO₂,step-wise gradient from 2-20% MeOH in CH₂Cl₂) to give 2 (2.08 g, 96%) asa clear oil.

O-Propargyl Boc-Tyrosine N-Hydroxysuccinimide Ester (3)

Disuccinimidyl carbonate (2.46 g, 9.60 mmol) was added to a solution of2 (2.04 g, 6.40 mmol) and pyridine (1.04 mL, 12.80 mmol) in acetonitrile(16 ml). The reaction was stirred at room temperature for 15 h, duringwhich the solution became clear and evolved gas. The reaction mixturewas added to EtOAc, washed twice with 1 N HCl and twice with saturatedNaHCO₃, dried over Na₂SO₄, and concentrated in vacuo to yield a whitesolid (1.90 g, 71%), which was used in the next step without furtherpurification.

tert-Butyl((S)-1-(((2S,3S,4R,5R)-5-(6-(dimethylamino)-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl)amino)-1-oxo-3-(4-(prop-2-yn-1-yloxy)phenyl)propan-2-yl)carbamate(O-propargyl Boc-puromycin) (4)

O-Propargyl Boc-Tyrosine N-hydroxysuccinimide ester 3 (1.42 g, 3.40mmol) and triethylamine (0.47 mL, 3.40 mmol) were added to a solution ofpuromycin aminoglycoside (500.0 mg, 1.70 mmol) in CH₂Cl₂ (15 mL). Thesolution was stirred at room temperature for 1.5 h and then directlypurified by flash chromatography (SiO₂, step-wise gradient from 2-10%MeOH in CH₂Cl₂), to yield the product as a white solid (480 mg, 47%). ¹HNMR (600 MHz, DMSO-d₆): δ 8.44 (s, 1H), 8.24 (s, 1H), 8.00 (d, J=7.8 Hz,1H), 7.21 (d, J=8.4 Hz, 2H), 6.84-6.92 (m, 3H), 6.05 (d, J=4.8 Hz, 1H),5.99 (d, J=3.0 Hz, 1H), 5.15 (t, J=5.4 Hz, 1H), 4.74 (d, J=1.8 Hz, 2H),4.43-4.53 (m, 2H), 4.17-4.24 (m, 1H), 3.91-3.96 (m, 1H), 3.64-3.72 (m,1H), 3.10-3.62 (m, 8H), 2.91 (dd, J=13.8, 4.2 Hz, 1H), 2.70 (dd, J=13.8,10.2 Hz, 1H), 1.30 (s, 9H); LC/MS (ESI, m/z): calcd for C₂₉H₃₇N₇O₇[M+H]⁺ 596. found 596.

(S)-2-Amino-N-((2S,3S,4R,5R)-5-(6-(dimethylamino)-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl)-3-(4-(prop-2-yn-1-yloxy)phenyl)propanamide(O-propargyl-puromycin) (5)

O-Propargyl Boc-Puromycin 4 (480 mg, 0.81 mmol) was dissolved in a 1:1TFA (4 mL) and CH₂Cl₂ (4 mL) mixture and then stirred at roomtemperature for 30 min. Volatiles were evaporated in vacuo and theresidue was dissolved in CH₂Cl₂. The solution was poured into saturatedaqueous NaHCO₃. The organic layer was dried over Na₂SO₄ and evaporatedto dryness in vacuo. The residue was purified by flash chromatography(SiO₂, step-wise gradient from 5-15% MeOH in CH₂Cl₂) to afford theO-propargyl-puromycin 5 (111 mg, 28%) as a white solid. ¹H NMR (600 MHz,DMSO-d₆): δ 8.44 (s, 1H), 8.24 (s, 1H), 8.06 (br, 1H), 7.17 (d, J=8.4Hz, 2H), 6.90 (d, J=8.4 Hz, 2H), 6.13 (d, J=5.4 Hz, 1H), 5.98 (d, J=3.0Hz, 1H), 5.13 (t, J=5.4 Hz, 1H), 4.74 (d, J=1.8 Hz, 2H), 4.42-4.51 (m,2H), 3.91-3.96 (m, 1H), 3.66-3.72 (m, 1H), 3.26-3.58 (m, 9H), 2.92 (dd,J=14.4, 4.8 Hz, 1H), 2.52-2.58 (m, 1H), 2.02 (br, 2H); ¹³C NMR (150 MHz,DMSO-d₆): δ 174.6, 155.7, 154.3, 151.8, 149.6, 137.9, 131.3, 130.2,119.6, 114.5, 89.4, 83.5, 79.4, 78.0, 73.2, 61.0, 56.1, 55.3, 50.0,40.4, 40.0; HRMS: (ESI, m/z) calcd [M+H]⁺ for C₂₄H₂₉N₇O₅: 496.2303.found 496.2308.

Example 2 OP-Puro Labeling of Cultured Cells and Detection byFluorescence Microscopy

A chemically-tagged puro was developed to label newly synthesizedproteins, for subsequent imaging by fluorescence microscopy and forisolation by affinity chromatography. Structure-activity studies of puro(Nathans et al. Nature 197:1076-1077 (1963); Pestka et al. Antimicrob.Agents. Chemother. 4(1):37-43 (1973); Eckermann et al. Eur. J. Biochem.41(3):547-554 (1974); Vanin et al. FEBS Lett. 40(1):124-126 (1974); Leeet al. J. Med. Chem. 24(3):304-308 (1981)) indicated that the moleculetolerates modifications of the O-Me phenyl ring, without significantloss of activity. O-propargyl-puromycin (OP-puro, FIG. 1B), a puroanalog that bears a terminal alkyne group, was synthesized to allowdetection of nascent polypeptide chains by copper(I)-catalyzedazide-alkyne cycloaddition (CuAAC) (Wang et al., J. Am. Chem. Soc.125(11):3192-3193).

NIH-3T3 cells were grown on glass coverslips in Dulbecco modifiedEagle's medium (DMEM) supplemented with 10% bovine calf serum,penicillin and streptomycin. OP-puro was added to cells in completeculture medium. After incubation for the desired amount of time, thecells were washed with PBS and then fixed with cold methanol for 2minutes at −20° C. The cells were washed with TBS (10 mM Tris pH 7.5,150 mM NaCl), permeabilized with TBST (TBS with 0.2% Triton X-100), andthen washed with TBS again. CuAAC detection of OP-puro incorporated intonascent protein was performed by reacting the fixed cells for 30 minutesat room temperature with 20 μM Alexa568-azide, as described (Salic etal., Proc. Natl. Acad. Sci. USA 105(7):2415-2420 (2008)). Cu(I) wasgenerated in situ from CuSO4 and ascorbic acid. After staining, thecoverslips were washed several times with TBST, counterstained withHoechst, and mounted in standard mounting media. The stained cells wereimaged by DIC and by epi-fluorescence microscopy on a Nikon TE2000Umicroscope equipped with an OrcaER camera (Hammamatsu), and 20× PlanApo0.75 NA and 40× PlanApo 0.95 NA air objectives (Nikon). Images werecollected using Metamorph image acquisition software (AppliedPrecision).

To determine if protein synthesis is required for OP-puro incorporationinto nascent polypeptides, cells were incubated with OP-puro, in thepresence or absence of 50 micrograms/mL cycloheximide (CHX), added tothe cells 15 minutes before OP-puro. This concentration of CHX wasdetermined to completely block protein translation in cells.

When cultured cells were treated for one hour with varyingconcentrations of OP-puro followed by fixation and staining withAlexa568-azide, a specific fluorescent signal proportional to theconcentration of OP-puro was detected (FIG. 2A). OP-puro incorporationrequired functional ribosomes and was abolished if cells were treatedwith CHX (FIG. 2A, right-most panel), as seen before by autoradiography(FIG. 1E). The intensity of the OP-puro stain increased with incubationtime (FIG. 2B) and reached saturation after about one hour. A strongsignal was seen after as little as 15 minutes of incubation withOP-puro. While the OP-puro staining pattern was mostly cytoplasmic, manycells also showed a punctate nuclear stain, suggesting that thetruncated protein-OP-puro conjugates released from ribosomes canlocalize to various subcellular compartments. At later time points (seethe 3 hour image of FIG. 2B), the cytoplasmic OP-puro signal issignificantly decreased, suggesting that the polypeptide-OP-puroconjugates are turned over.

The OP-puro conjugates do not form if protein synthesis is inhibited byCHX, which blocks translational initiation, demonstrating thattranslating ribosomes are required for OP-puro incorporation intonascent polypeptide chains, at the level of polypeptide chainelongation. These results demonstrate that OP-puro is a translationinhibitor that forms covalent adducts with elongating polypeptide chainson the ribosome. A concentration of 25 microM OP-puro blocked proteinsynthesis almost completely, defining a concentration that shouldcapture almost quantitatively the proteins synthesized by a given cellin the form of OP-puro-polypeptide conjugates.

To determine the effect of inhibiting the proteasome on the stability ofOP-puro-conjugated polypeptide chains, cells were pulse-labeled with 50microM OP-puro for 15 minutes and were then incubated in complete mediasupplemented with 50 micrograms/mL CHX (to block further incorporationof OP-puro into nascent proteins), in the presence or absence of 5microM of the proteasome inhibitor bortezomib. Cells were fixed at theindicated times and were stained in parallel with Alexa568-azide. Twonegative controls were used: 1) untreated cells; and 2) cellspre-incubated with 50 micrograms/mL CHX for 15 minutes, followed byincubation with 50 microM OP-puro and 50 micrograms/mL CHX for another15 minutes.

Polypeptide-puro conjugates have been reported to be unstable (Goldberg,Proc. Natl. Acad. Sci. USA 69(2):422-426 (1972); Wharton et al. FEBSLett. 168(1):134-138 (1984)). Cultured cells were labeled with a shortpulse of OP-puro, after which the cells were washed and chased in theabsence or presence of the proteasome inhibitor bortezomib (Adams et al.Cancer Invest. 22(2):304-311 (2004)). In the absence of bortezomib,OP-puro conjugates are unstable and disappear from cells in under 1 hour(FIG. 2C, middle panel). If the proteasome is inhibited with bortezomib,the OP-puro conjugates become stable (FIG. 2C, bottom row of panels),demonstrating that the rapid disappearance of OP-puro conjugates is dueto degradation by the proteasome. We conclude that the majority ofconjugates of OP-puro with nascent polypeptide chains are short-lived,which suggests that the OP-puro signal is a good reflection of instantprotein synthesis in cells.

Example 3 Inhibition of Translation by OP-Puro

A plasmid carrying a GFP fusion of the mouse Suppressor of Fused (SuFu)gene under the control of an SP6 RNA polymerase promoter was used togenerate 35[S]-Met-labeled GFP-SuFu by in vitro translation in rabbitreticulocyte lysates (TNT SP6 Quick coupled transcription/translationkit, Promega), according to the manufacturer's instructions. Translationreactions were performed for 1 hour at 30° C., in the absence orpresence of varying concentrations of puro and OP-puro. The reactionswere stopped by addition of SDS-PAGE sample buffer with 50 mM DTT andboiling. Equal amounts of lysate were separated by SDS-PAGE and theamount of in vitro translated GFP-SuFu was determined byautoradiography.

To measure translation inhibition in cells, human embryonic kidney 293Tcells were pre-incubated for 30 minutes with varying concentrations ofpuro or OP-puro, in complete media. The cells were then incubated for 3hours in Met-free media supplemented with 35[S]-Met (from Perkin-Elmer,at 100 microCi/mL final concentration), in the continued presence ofvarying concentrations of puro or OP-puro. The cells were harvested,lyzed in TBS with 1% Triton X-100 and protease inhibitors (Completetablets, Roche), and centrifuged for 15 minutes at 20,000 g in arefrigerated centrifuge. The clarified cells lysates were analyzed bySDS-PAGE, followed by autoradiography, to measure bulk proteintranslation.

OP-puro inhibits protein synthesis, both in reticulocyte lysates (FIG.1C) and in cultured cells (FIG. 1D), displaying a potency 2-3 fold lowerthan that of unmodified puro.

Example 4 Affinity Purification of Nascent Polypeptide-OP-PuroConjugates

Human 293T cells were labeled for 1 hour in Met-free DMEM supplementedwith 10% dialyzed fetal bovine serum and 35[S]-Met (100 microCi/mLfinal). The cells were then incubated in the same media, in the presenceof 10 microM puro, 25 microM OP-puro or 25 microM OP-puro and 50micrograms/mL CHX, for an additional 1 hour. The cells were harvested,washed with ice cold PBS and then lyzed on ice in 100 mM Tris pH 8.5with 1% Triton-X10 and protease inhibitors. The lysates were clarifiedby centrifugation for 15 minutes at 20,000 g and 4° C., and were thensubjected to CuAAC with biotin azide for 30 minutes at room temperature.The final concentrations in the CuAAC reaction were: 100 microM biotinazide, 1 mM CuSO₄ and 50 mM ascorbic acid (added last to the lysates).Biotinylated proteins were diluted in binding buffer (20 mM Tris pH 8,500 mM NaCl, 1% Triton-X100) and were bound to Neutravidin beads(Pierce). After extensive washes with binding buffer, bound proteinswere eluted, separated by SDS-PAGE and detected by autoradiography.OP-puro formed conjugates with 35[S]-Met-labeled nascent polypeptidechains (FIG. 1E), which can be specifically retrieved on streptavidinbeads.

Example 5 Use of OP-Puro to Image Protein Synthesis in Animals

One hundred microliters of a 20 mM solution of OP-puro in PBS wereinjected intraperitoneally into a 3-week old mouse, while a mouseinjected with 100 microliters of PBS was used as negative control.Various organs were harvested after one hour and were fixed in formalinovernight. Organ fragments were embedded in paraffin, sectioned, andwashed with xylene to remove the paraffin. After washing with ethanoland rehydration in TBS, the tissue sections were stained with 20 microMtetramethylrhodamine (TMR)-azide, as described (Salic et al., Proc.Natl. Acad. Sci. USA 105(7):2415-2420 (2008)). The tissue sections werecounterstained with Hoechst, mounted in standard mounting media and werethen imaged by fluorescence microscopy and DIC.

Mice were injected intraperitoneally with OP-puro, and tissues wereharvested 1 hour later, fixed and stained with fluorescent azide, eitherafter sectioning or in whole mount. As shown in FIGS. 3 and 4, tissuesfrom uninjected mice showed low non-specific staining, while tissuesfrom OP-puro-injected mice displayed specific patterns of OP-puroincorporation into nascent proteins. In the small intestine, translationwas strongest in cells in the crypts and at the base of intestinal villi(FIG. 3A), consistent with the high proliferative and secretory activityof these cells. The stain was particularly strong in Paneth cells, whichare located close to the base of the crypts and are filled withsecretory vesicles. The intense OP-puro labeling of vesicles in Panethcells (FIG. 3A, bottom panels) suggests that prematurely-terminated,OP-puro-conjugated secretory proteins are translocated into the ERlumen, as described before for puro (Andrews et al., Biochem. J.121(4):683-694 (1971)). The same pattern of OP-puro labeling wasobserved in whole mount stains of the small intestine (FIG. 3B),suggesting that OP-puro is uniquely suited for visualizing proteinsynthesis in whole tissues and organs, with high sensitivity.

Patterns of protein synthesis in other mouse tissues surveyed (liver,kidney, and spleen) are shown in FIG. 4. The level of protein synthesisvaries between tissues, being the strongest in hepatocytes (FIG. 4A),consistent with the high levels of protein synthesis in the liver.Protein synthesis levels are uniformly high in hepatocytes but can varysignificantly within other tissues, such as spleen (FIG. 4C), in whichthe strongest OP-puro signal is found at the organ's periphery, underthe capsule. Interestingly, in muscle the OP-puro stain shows a strikingstriated pattern (FIG. 3C), suggesting that some muscle OP-puro-proteinconjugates are properly incorporated into sarcomeres, This method mightthus be suitable for imaging the assembly and turnover of structuressuch as sarcomeres.

Example 6 Preparation of Azido-Puromycin

(S)-Methyl3-(4-(2-bromoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate(1A)

2-Bromo-ethanol (0.61 mL, 8.03 mmol), followed by PPh₃ (3.16 g, 12.05mmol) was added to a solution of Boc-Tyr-OMe (2.85 g, 9.64 mmol) inanhydrous THF (50 mL). The mixture was cooled to 0° C. and DEAD (5.5 mLof 40% wt solution in toluene, 12.05 mmol) was added. The mixture wasallowed to warm at room temperature and was stirred overnight. After thereaction was finished, the solvent was evaporated and the residuedissolved in EtOAc (100 mL) was washed with NaOH (0.1 M, 2×50 mL),followed by brine. The organic phase was dried over Na₂SO₄ andevaporated. The crude product was purified by flash columnchromatography (SiO₂, step-wise gradient from 2-40% EtOAc in Hexanes) togive 1A (1.61 g, 50%) as a white solid. ¹H NMR (600 MHz, CDCl₃): δ 7.04(2H, d, J=8.4 Hz), 6.84 (2H, d, J=8.4 Hz), 4.96 (1H, d, J=7.8 Hz),4.51-4.58 (1H, m), 4.27 (2H, t, J=6.6 Hz), 3.71 (3H, s), 3.62 (2H, t,J=6.6 Hz), 3.06 (1H, dd, J=13.8, 5.4 Hz), 3.00 (1H, dd, J=13.8, 5.4 Hz),1.42 (9H, s); ¹³C NMR (150 MHz, CDCl₃): δ 172.3, 157.2, 155.1, 130.4,128.8, 114.8, 79.9, 67.9, 54.5, 52.2, 37.5, 29.1, 28.3; MS (ESI, m/z):424 (M+Na)⁺.

(S)-Methyl3-(4-(2-azidoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate(2A)

A mixture of 1A (1.61 g, 4.0 mmol) and sodium azide (0.78 g, 12.0 mmol)in DMF (27 mL) was stirred at room temperature for 20 h. The reactionmixture was diluted with H₂O (120 mL) and extracted with EtOAc (3×60mL). The combined organic phases were washed with brine, dried overNa₂SO₄ and concentrated in vacuo. The residue was purified by flashcolumn chromatography (SiO₂, step-wise gradient from 2-40% EtOAc inHexanes) to give 2A (1.46 g, 100%) as a clear oil. ¹H NMR (600 MHz,CDCl₃): δ 7.05 (2H, d, J=8.4 Hz), 6.85 (2H, d, J=8.4 Hz), 4.97 (1H, d,J=7.8 Hz), 4.51-4.58 (1H, m), 4.13 (2H, t, J=5.4 Hz), 3.71 (3H, s), 3.58(2H, t, J=5.4 Hz), 3.06 (1H, dd, J=13.8, 5.4 Hz), 3.00 (1H, dd, J=13.8,5.4 Hz), 1.42 (9H, s); ¹³C NMR (150 MHz, CDCl₃): δ 172.3, 157.3, 155.1,130.4, 128.7, 114.6, 79.9, 66.9, 54.5, 52.2, 50.2, 37.5, 28.3; MS (ESI,m/z): 387 (M+Na)⁺.

(S)-3-(4-(2-Azidoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoicacid (3A)

A solution of 2A (1.46 g, 4.0 mmol) was dissolved in 1,2-dichloroethane(20 mL) and after addition of trimethyltin hydroxide (2.17 g, 12.0mmol), the mixture was heated at 80° C. until TLC analysis indicated acomplete reaction. After completion of the reaction, the mixture wasconcentrated in vacuo, and the residue was taken up in EtOAc (80 mL).The organic layer was washed with aqueous HCl (5%) (3×50 mL). Theorganic layer was then washed with brine and dried over Na₂SO₄. Afterremoval of the solvent in vacuo, the residue was purified by flashcolumn chromatography (SiO₂, step-wise gradient from 2-20% MeOH inCH₂Cl₂) to give 3A (0.98 g, 70%) as a white solid. ¹H NMR (600 MHz,CDCl₃): δ 7.11 (2H, d, J=8.4 Hz), 6.86 (2H, d, J=8.4 Hz), 4.94 (1H, d,J=7.2 Hz), 4.53-4.60 (1H, m), 4.13 (2H, t, J=5.4 Hz), 3.58 (2H, t, J=5.4Hz), 3.14 (1H, dd, J=13.8, 5.4 Hz), 3.04 (1H, dd, J=13.8, 5.4 Hz), 1.43(9H, s); ¹³C NMR (150 MHz, CDCl₃): δ 157.3, 155.5, 130.5, 128.8, 114.7,80.2, 66.9, 54.7, 50.1, 37.0, 28.3; MS (ESI, m/z): 373 (M+Na)⁺.

tert-Butyl((S)-3-(4-(2-azidoethoxy)phenyl)-1-(((2S,3S,4R,5R)-5-(6-(dimethylamino)-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl)amino)-1-oxopropan-2-yl)carbamate(Boc-Puromycin Analog) (4A)

3A (289 mg, 0.82 mmol) and puromycin aminoglycoside (221 mg, 0.75 mmol)was dissolved in dry DMF (6 mL), followed by HATU (314 mg, 0.82 mmol)and diisopropylethylamine (0.26 mL, 1.5 mmol). The reaction mixture wasthen stirred at room temperature for 3 h, then diluted with H₂O (80 mL)and saturated NaHCO₃ (40 mL), extracted with EtOAc (3×40 mL). Thecombined organic phases were washed with brine, dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by flash chromatography(SiO₂, step-wise gradient from 2-15% MeOH in CH₂Cl₂) to afford 4A (384mg, 82%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆): δ 8.45 (1H, s),8.24 (1H, s), 8.00 (1H, d, J=7.8 Hz), 7.21 (2H, d, J=8.4 Hz), 6.84-6.91(3H, m), 6.06 (1H, d, J=4.8 Hz), 6.00 (1H, d, J=3.0 Hz), 5.16 (1H, t,J=5.4 Hz), 4.44-4.54 (2H, m), 4.21 (1H, dt, J=9.6, 4.8 Hz), 4.13 (2H, t,J=5.4 Hz), 3.92-3.98 (1H, m), 3.10-3.80 (10H, m), 2.92 (1H, dd, J=13.8,4.8 Hz), 2.70 (1H, dd, J=13.8, 9.6 Hz), 1.31 (9H, s); ¹³C NMR (150 MHz,DMSO-d₆): δ 172.0, 156.5, 155.2, 154.3, 151.8, 149.7, 137.9, 130.5,130.3, 119.6, 114.1, 89.3, 83.4, 78.0, 73.1, 66.7, 60.9, 55.9, 50.3,49.6, 36.9, 28.1, 27.8; MS (ESI, m/z): 627 (M+H)⁺.

(S)-2-Amino-3-(4-(2-azidoethoxy)phenyl)-N-(((2S,3S,4R,5R)-5-(6-(dimethylamino)-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl)propanamide(Azidoethyl-Puromycin, AE-Puro) (5A)

Boc-Puromycin analog 4A (392 mg, 0.63 mmol) was dissolved in a 1:1 TFA(5 mL) and CH₂Cl₂ (5 mL) mixture and then stirred at room temperaturefor 30 min. Volatiles were evaporated in vacuo and the residue wasdissolved into CH₂Cl₂. The solution was poured into a suspension ofsaturated aqueous NaHCO₃. The organic layer was dried over Na₂SO₄ andevaporated to dryness in vacuo. The residue was purified by flashchromatography (SiO₂, step-wise gradient from 2-15% MeOH in CH₂Cl₂) toafford Puromycin analog 5A (151 mg, 46%) as a white solid. ¹H NMR (600MHz, DMSO-d₆): δ 8.45 (1H, s), 8.24 (1H, s), 8.06 (1H, br), 7.17 (2H, d,J=8.4 Hz), 6.87 (1H, d, J=8.4 Hz), 6.14 (1H, d, J=4.8 Hz), 5.98 (1H, d,J=2.4 Hz), 5.14 (1H, t, J=5.4 Hz), 4.42-4.52 (2H, m), 4.14 (2H, t, J=5.4Hz), 3.92-3.97 (1H, m), 3.10-3.80 (12H, m), 2.92 (1H, dd, J=13.8, 5.4Hz), 2.55 (1H, dd, J=13.8, 9.0 Hz), 1.72 (2H, br); ¹³C NMR (150 MHz,DMSO-d₆): δ 174.8, 156.5, 154.3, 151.8, 149.6, 137.9, 131.1, 130.3,119.6, 114.2, 89.4, 83.6, 79.1, 73.2, 66.7, 61.0, 56.3, 50.0, 49.6,40.1; MS (ESI, m/z): 527 (M+H)⁺.

Example 7 Inhibition of Translation by AE-Puro

A plasmid carrying a GFP fusion of the mouse Suppressor of Fused (SuFu)gene under the control of an SP6 RNA polymerase promoter was used togenerate ³⁵S-Met-labeled GFP-SuFu by in vitro translation in rabbitreticulocyte lysates (TNT SP6 Quick coupled transcription/translationkit, Promega), according to the manufacturer's instructions. Translationreactions were performed for 1 hour at 30° C., in the absence orpresence of varying concentrations of puro and AE-puro (azido-puromycin,FIG. 5A). The reactions were stopped by addition of SDS-PAGE samplebuffer with 50 mM DTT and boiling. Equal amounts of lysate wereseparated by SDS-PAGE and the amount of in vitro translated GFP-SuFu wasdetermined by autoradiography. AE-puro inhibits protein synthesis inreticulocyte lysates (FIG. 5B) with a potency equal to that ofunmodified puromycin.

OTHER EMBODIMENTS

This application refers to various issued patents, published patentapplications, journal articles, books, manuals, and other publications,all of which are incorporated herein by reference.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

1-161. (canceled)
 162. A labeled polypeptide comprising a puromycin analog conjugate of the following formula:

wherein: R^(A) is a bond, or an optionally substituted aliphatic group, heteroaliphatic group, aryl group, and heteroaryl group, or a combination thereof; R^(B) is a bond or a C₁₋₆ aliphatic moiety; R¹ is hydrogen or a reactive group capable of undergoing a bioorthogonal reaction; R^(1′) is hydrogen or a reactive group capable of undergoing a bioorthogonal reaction; wherein R1 and R1′ are not simultaneously hydrogen; R² is hydrogen or C₁₋₆ aliphatic group; R³ and R⁴ are each independently hydrogen or a protecting group; and R⁶ is hydrogen or C₁₋₆ aliphatic group; or a salt thereof.
 163. The labeled polypeptide according to claim 162, wherein the puromycin analog conjugate is of the following formula:

or a salt thereof.
 164. The labeled polypeptide according to claim 162, wherein R^(A) is:


165. The labeled polypeptide according to claim 162, wherein the —R^(A)—R¹ group is selected from the group consisting of:


166. The labeled polypeptide according to claim 162, wherein R^(A) is an -alkylaryl group, a tyrosine side chain, or an alkyl group (e.g. a C₁₋₃ alkyl group).
 167. The labeled polypeptide according to claim 162, wherein R¹ comprises a dipolarophile.
 168. The labeled polypeptide according to claim 162, wherein R¹ comprises an ethynyl or a propargyl group.
 169. The labeled polypeptide according to claim 162, wherein R¹ comprises a 1,3-dipole.
 170. The labeled polypeptide according to claim 162, wherein R¹ comprises an azido or an azidoethyl group.
 171. The labeled polypeptide according to claim 162, wherein R¹ is an aldehyde or a quadricyclane.
 172. The labeled polypeptide according to claim 162, wherein R¹ comprises a tetrazine or a trans-cyclooctene.
 173. The labeled polypeptide according to claim 162, wherein the bioorthogonal reaction is a Staudinger ligation, a tetrazine ligation, an oxime ligation, a hydrazone ligation, and a quadricyclane ligation.
 174. A labeled polypeptide prepared by the method comprising: providing a polypeptide-puromycin analog conjugate comprising a first reactive group; and contacting the polypeptide-puromycin analog conjugate with a compound comprising a second reactive group and a label, such that a bioorthogonal reaction occurs between the first and second reactive groups.
 175. The labeled polypeptide according to claim 174, wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.
 176. The labeled polypeptide according to claim 174, wherein the first and second reactive groups are capable of undergoing a [3+2] cycloaddition, a Staudinger ligation, an inverse electron demand Diels-Alder reaction (e.g., tetrazine ligation), an oxime addition, a hydrazone addition, or a [2+2+2] cycloaddition (e.g., quadricyclane ligation).
 177. The labeled polypeptide according to claim 175, wherein the a 1,3-dipole is a nitrile oxide, an azide, a diazomethane, a nitrone, or a nitrile imine.
 178. The labeled polypeptide according to claim 175, wherein the dipolarophile is an alkene or an alkyne.
 179. The labeled polypeptide according to claim 174, wherein the first reactive group is an ethynyl or propargyl group and the second reactive group is an azido group.
 180. The labeled polypeptide according to claim 174, wherein the first reactive group is an azido group and the second reactive group is an ethynyl group or propargyl group.
 181. The labeled polypeptide according to claim 174, comprising a cycloadduct resulting from a [3+2] cycloaddition between an ethynyl group and an azido group.
 182. The labeled polypeptide according to claim 174, comprising a label that is directly detectable.
 183. The polypeptide according to claim 182, wherein the label is a fluorescent agent.
 184. The polypeptide according to claim 183, wherein the label is biotin.
 185. A cell comprising a labeled polypeptide according to claim
 162. 186. An organism comprising a labeled polypeptide according to claim
 162. 187. The organism according to claim 186, wherein the organism is a non-human whole animal.
 188. The labeled polypeptide according to claim 162, wherein the puromycin analog is of the formula:

or a salt thereof.
 189. A compound of the following formula:

or a salt thereof. 